Search

Supplementary Table 1 from Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Supplementary Table 2 from Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Supplementary Figure 1 from Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Supplementary Table 3 from Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Understanding molecular mechanisms underlying lung cancer is a prerequisite toward treatment. To enable mechanistic investigations into the epigenetic regulation of the tumor suppressor gene cell adhesion molecule 1 (Cadm1) in lung cancer progenitor cells, we developed 10 cell lines from single, spontaneously transformed lung tumor cells isolated from c-Myc and c-Raf double-transgenic mice. Specifically, we investigated Cadm1 promoter hypermethylation, which was significantly induced in transgenic transformed cells. Analysis of 69 CpGs displayed differential methylation pattern between and within progenitor cell lines, and the degree of methylation correlated well with transcriptional repression. Indeed, restoration of Cadm1 gene expression was achieved by treatment with the experimental demethylating drug 5-aza-2′-deoxycytidine. Furthermore, methylation of core CpGs in the binding sites of Sp1, Sp3, and zinc finger 5 along the promoter region of Cadm1 abrogated DNA-protein binding. Treatment with mithramycin A, an inhibitor of Sp1 or Sp3 binding, resulted in reduction of Cadm1 gene expression, therefore suggesting a potential role of Sp1/Sp3 in Cadm1 regulation. Identifying molecular rules for the epigenetic control of tumor suppressor genes enables mechanistic insights into lung cancer growth and opportunities for novel therapies. [Cancer Res 2008;68(18):7587–96]

ObjectiveHuman precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA 29 isolation from PCLS to enable miRNA readouts. Results The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human 38 PCLS model.

Cellular senescence is a stable cell cycle arrest induced by diverse triggers, including replicative exhaustion, DNA damaging agents, oncogene activation, oxidative stress, and chromatin disruption. With important roles in aging and tumor suppression, cellular senescence has been implicated also in tumor promotion. Here we show that certain multiwalled carbon nanotubes (MWCNTs), as fiber-like nanomaterials, can trigger cellular senescence in primary human mesothelial cells. Using in vitro approaches, we found manifestation of several markers of cellular senescence, especially after exposure to a long and straight MWCNT. These included inhibition of cell division, senescence-associated heterochromatin foci, senescence-associated distension of satellites, LMNB1 depletion, γH2A.X nuclear panstaining, and enlarged cells exhibiting senescence-associated β-galactosidase activity. Furthermore, genome-wide transcriptome analysis revealed many differentially expressed genes, a mong which were genes encoding for a senescence-associated secretory phenotype. Our results clearly demonstrate the potential of long and straight MWCNTs to induce premature cellular senescence. This finding may find relevance in risk assessment of workplace safety, and in evaluating MWCNT's use in medicine such as drug carrier, due to exposure effects that might prompt onset of age-related diseases, or even carcinogenesis.

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.

Increasing evidences point to a functional unit from mast cell and sensory nerve as a key component of pathophysiological responses. Here we propose a subpopulation of neurokinin-1 receptor (NK1R) expressing mast cells in the peripheral tissue of humans to be involved in airway hyper responsivness (AHR).Human precision cut lung slices PCLS were generated using resection material from lung tumor patients and passivly sensitized with plasma from HDM-allergic patients. Tissue was treated with 10 µM histamine 1 receptor (H1R) or 100µM NK1R antagonists or a monoclonal antibody against IgE (mab IgE) during sensitization. Subsequently, bronchoconstriction was provoked with the sensory nerve agonist capsaicin. Bronchoconstriction was measured by video microscopy. The reduction of the airway area in percentage of initial airway area was analysed. Mast cell tryptase, IgE and NK1R immunostaining was performed in PCLS in order to visualize mast cell subpopulations. Immunostaining of sensory nerve fibers and mast cells showed anatomical proximity in the airways. A subpopulation of NK1 receptor positive mast cells were identified. After passive sensitization, capsaicin‑induced bronchoconstriction showed reduction of the airway area of 40%. Capsaicin‑induced bronchoconstriction was completely inhibited by pre-incubation with NK1R and H1R antagonists or mab IgE. Capsaicin-induced significant increase in bronchoconstriction in passive sensitized human PCLS. Capsaicin induced bronchoconstriction under asthmatic conditions is mediated by a process involving a subpopulation of NK1R positive mast cells.

Background Recombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome. Results We analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector. Conclusions Our results suggest that the chromatin state along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0238-0) contains supplementary material, which is available to authorized users.

Congenital heart disease (CHD) is among the most prevalent and fatal of all birth defects. Deciphering its causes, however, is complicated, as many patients affected by CHD have no family history of the disease. There is also widespread heterogeneity of cardiac malformations within affected individuals. Nonetheless, there have been tremendous efforts toward a better understanding of the molecular and cellular events leading to CHD. Notably, certain cardiac-specific transcription factors have been implicated in mammalian heart development and disruption of their activity has been demonstrated in CHD. The homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. Thus, NKX2-5 appears to be hypermutable, yet the overall detection frequency in sporadic CHD is about 2% and NKX2-5 mutations are one-time detections with single-positives or private to families. Furthermore, there is lack of genotype-phenotype correlation, in which the same cardiac malformations have been exhibited in different NKX2-5 mutations or the same NKX2-5 mutation associated with diverse malformations. Here, we summarize published NKX2-5 germline mutations and explore different avenues in disease pathogenesis to support the notion of a multifactorial cause of CHD where possibly several genes and associated pathways are involved.

'Epigenetics' is a heritable phenomenon without change in primary DNA sequence. In recent years, this field has attracted much attention as more epigenetic controls of gene activities are being discovered. Such epigenetic controls ensue from an interplay of DNA methylation, histone modifications, and RNA-mediated pathways from non-coding RNAs, notably silencing RNA (siRNA) and microRNA (miRNA). Although epigenetic regulation is inherent to normal development and differentiation, this can be misdirected leading to a number of diseases including cancer. All the same, many of the processes can be reversed offering a hope for epigenetic therapies such as inhibitors of enzymes controlling epigenetic modifications, specifically DNA methyltransferases, histone deacetylases, and RNAi therapeutics. 'In utero' or early life exposures to dietary and environmental exposures can have a profound effect on our epigenetic code, the so-called 'epigenome', resulting in birth defects and diseases developed later in life. Indeed, examples are accumulating in which environmental exposures can be attributed to epigenetic causes, an encouraging edge towards greater understanding of the contribution of epigenetic influences of environmental exposures. Routine analysis of epigenetic modifications as part of the mechanisms of action of environmental contaminants is in order. There is, however, an explosion of research in the field of epigenetics and to keep abreast of these developments could be a challenge. In this paper, we provide an overview of epigenetic mechanisms focusing on recent reviews and studies to serve as an entry point into the realm of 'environmental epigenetics'.

Poor maternal nutrition during gestation can lead to intrauterine growth retardation (IUGR), a main cause of low birth weight associated with high neonatal morbidity and mortality. Such early uterine environmental exposures can impact the neonatal epigenome to render later-in-life disease susceptibility. We established in Wistar Han rats a mild IUGR model induced by gestational protein deficiency (i.e. 9% crude protein in low protein diet vs. 21% in control, from GD 0 to 21) to identify alterations in gene expression and methylation patterns in certain genes implicated in human IUGR or in placental development. We found differential gene expression of Wnt2 and Dlk1 between IUGR and control. Notably, Wnt2 exhibited significant decrease while Dlk1 increase in IUGR placentas, correlating to decrease in fetal and placental weight. Methylation patterns encompassing 30 CpGs in the Wnt2 promoter region revealed variability in both IUGR and control placentas, but a site-specific hypomethylation was evident in IUGR placentas. Our present findings further support a key role of maternal gestational nutrition in defining the neonatal epigenome.

Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and ‘bivalent’ histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

Heart and neural crest derivatives expressed 1 (HAND1) is a basic helix-loop-helix (bHLH) transcription factor essential for mammalian heart development. Absence of Hand1 in mice results in embryonal lethality, as well as in a wide spectrum of cardiac abnormalities including failed cardiac looping, defective chamber septation and impaired ventricular development. Therefore, Hand1 is a strong candidate for the many cardiac malformations observed in human congenital heart disease (CHD). Recently, we identified a loss-of-function frameshift mutation (p.A126fs) in the bHLH domain of HAND1 frequent in hypoplastic hearts. This finding prompted us to continue our search for HAND1 gene mutations in a different cohort of malformed hearts affected primarily by septation defects. Indeed, in tissue samples of septal defects, we detected 32 sequence alterations leading to amino acid change, of which 12 are in the bHLH domain of HAND1. Interestingly, 10 sequence alterations, such as p.L28H and p.L138P, had been identified earlier in hypoplastic hearts, but the frequent p.A126fs mutation was absent except in one aborted case with ventricular septal defect and outflow tract abnormalities. Functional studies in yeast and mammalian cells enabled translation of sequence alterations to HAND1 transcriptional activity, which was reduced or abolished by certain mutations, notably p.L138P. Our results suggest that HAND1 may also be affected in septation defects of the human hearts, and thus has a broader role in human heart development and CHD.

Toxicogenomics is a field that emerged from the combination of conventional toxicology with functional genomics. In recent years, this field contributed immensely in defining adverse biological effects resulting from environmental stressors, toxins, drugs and chemicals. Through microarray technology, large-scale detection and quantification of mRNA transcripts and of microRNAs, related to alterations in mRNA stability or gene regulation became feasible. Other 'omics' technologies, notably proteomics and metabonomics soon joined in providing further fine tuning in the gathering and interpretation of toxicological data. A field that will inevitably modify the landscape for toxicogenomics is 'epigenetics', a term which refers to heritable changes in gene expression without accompanying alterations in the DNA sequence. These epigenetic changes are brought about by mechanisms such as DNA methylation, histone modifications, and non-coding RNAs in the regulation of gene expression patterns. Epigenetic mechanisms are essential in normal development and differentiation, but these can be misdirected leading to diseases, notably cancer. Indeed, there is now a mounting body of evidence that environmental exposures particularly in early development can induce epigenetic changes, which may be transmitted in subsequent generations or serve as basis of diseases developed later in life. In either way, epigenetic mechanisms will help interpret toxicological data or toxicogenomic approaches to identify epigenetic effects of environmental exposures. Thus, a full understanding of environmental interactions with the genome requires keeping abreast of epigenetic mechanisms, as well as conducting routine analysis of epigenetic modifications as part of the mechanism of actions of environmental exposure. A number of approaches are currently available to study epigenetic modifications in a gene-specific or genome-wide manner. Here we describe our approaches in studying the epigenetic modification of the tumor-suppressor gene Tslc1 (Igsf4a) in lung tumors obtained from transgenic mouse models.

Background The 3'-untranslated region (3'-UTR) of mRNA contains regulatory elements that are essential for the appropriate expression of many genes. These regulatory elements are involved in the control of nuclear transport, polyadenylation status, subcellular targetting as well as rates of translation and degradation of mRNA. Indeed, 3'-UTR mutations have been associated with disease, but frequently this region is not analyzed. To gain insights into congenital heart disease (CHD), we have been analyzing cardiac-specific transcription factor genes, including GATA4, which encodes a zinc finger transcription factor. Germline mutations in the coding region of GATA4 have been associated with septation defects of the human heart, but mutations are rather rare. Previously, we identified 19 somatically-derived zinc finger mutations in diseased tissues of malformed hearts. We now continued our search in the 609 bp 3'-UTR region of GATA4 to explore further molecular avenues leading to CHD. Methods By direct sequencing, we analyzed the 3'-UTR of GATA4 in DNA isolated from 68 formalin-fixed explanted hearts with complex cardiac malformations encompassing ventricular, atrial, and atrioventricular septal defects. We also analyzed blood samples of 12 patients with CHD and 100 unrelated healthy individuals. Results We identified germline and somatic mutations in the 3'-UTR of GATA4. In the malformed hearts, we found nine frequently occurring sequence alterations and six dbSNPs in the 3'-UTR region of GATA4. Seven of these mutations are predicted to affect RNA folding. We also found further five nonsynonymous mutations in exons 6 and 7 of GATA4. Except for the dbSNPs, analysis of tissue distal to the septation defect failed to detect sequence variations in the same donor, thus suggesting somatic origin and mosaicism of mutations. In a family, we observed c.+119A > T in the 3'-UTR associated with ASD type II. Conclusion Our results suggest that somatic GATA4 mutations in the 3'-UTR may provide an additional molecular rationale for CHD.

Background N-acetyltransferase 2 (NAT2) metabolizes arylamines and hydrazines moeities found in many therapeutic drugs, chemicals and carcinogens. The gene encoding NAT2 is polymorphic, thus resulting in rapid or slow acetylator phenotypes. The acetylator status may, therefore, predispose drug-induced toxicities and cancer risks, such as bladder, colon and lung cancer. Indeed, some studies demonstrate a positive association between NAT2 rapid acetylator phenotype and colon cancer, but results are inconsistent. The role of NAT2 acetylation status in lung cancer is likewise unclear, in which both the rapid and slow acetylator genotypes have been associated with disease. Methods We investigated three genetic variations, c.481C>T, c.590G>A (p.R197Q) and c.857G>A (p.G286E), of the NAT2 gene, which are known to result in a slow acetylator phenotype. Using validated PCR-RFLP assays, we genotyped 243 healthy unrelated Caucasian control subjects, 92 colon and 67 lung cancer patients for these genetic variations. As there is a recent meta-analysis of NAT2 studies on colon cancer (unlike in lung cancer), we have also undertaken a systematic review of NAT2 studies on lung cancer, and we incorporated our results in a meta-analysis consisting of 16 studies, 3,865 lung cancer patients and 6,077 control subjects. Results We did not obtain statistically significant differences in NAT2 allele and genotype frequencies in colon cancer patients and control group. Certain genotypes, however, such as [c.590AA+c.857GA] and [c.590GA+c.857GA] were absent among the colon cancer patients. Similarly, allele frequencies in lung cancer patients and controls did not differ significantly. Nevertheless, there was a significant increase of genotypes [c.590GA] and [c.481CT+c.590GA], but absence of homozygous c.590AA and [c.590AA+c.857GA] in the lung cancer group. Meta-analysis of 16 NAT2 studies on lung cancer did not evidence an overall association of the rapid or slow acetylator status to lung cancer. Similarly, the summary odds ratios obtained with stratified meta-analysis based on ethnicity, and smoking status were not significant. Conclusion Our study failed to show an overall association of NAT2 genotypes to either colon or lung cancer risk.

Birth defects are the leading cause of infant mortality and malformations in congenital heart disease (CHD) are among the most prevalent and fatal of all birth defects. Yet the molecular mechanisms leading to CHD are complex and the causes of the cardiac malformations observed in humans are still unclear. In recent years, the pivotal role of certain transcription factors in heart development has been demonstrated, and gene targeting of cardiac-specific transcription factor genes in animal models has provided valuable insights into heart anomalies. Nonetheless results in these models can be species specific, and in humans, germline mutations in transcription factor genes can only account for some cases of CHD. Furthermore, most patients do not have family history of CHD. There is, therefore, a need for a better understanding of the mechanisms in both normal cardiac development and the formation of malformations. The combining of expertise in cardiac anatomy, pathology, and molecular genetics is essential to adequately comprehend developmental abnormalities associated with CHD. To help elucidate genetic alterations in affected tissues of malformed hearts, we carried out genetic analysis of cardiac-specific transcription factor genes from the Leipzig collection of formalin-fixed malformed hearts. Working with this morphologically well-characterized archival material not only provided valuable genetic information associated with disease, but enabled us to put forward a hypothesis of somatic mutations as a novel molecular cause of CHD. Knowledge of cause and disease mechanism may allow for intervention that could modify the degree of cardiac malformations or development of new approaches for prevention of CHD.

Human heart development requires an orderly coordination of transcriptional programs, with the homeo-domainproteinNKX2-5beingoneofthekeytranscriptionfactorsrequiredforthedifferentiationofmesodermalprogenitor cells. Indeed, lack ofNkx2-5 in mice arrests heart development prior to looping, resulting inembryonic lethality. There are 28 germline NKX2-5 mutations identified in humans that are associated withcongenital heart disease, and we recently reported multiple somatic mutations in patients with complex car-diac malformations. To address the functional consequences of single and multiple mutations of NKX2-5,wedeveloped a functional assay in the budding yeast Saccharomyces cerevisiae, which could determine trans-activation capacity and specificity of expressedNKX2-5 alleles towards targeted response element (RE)sequences. We focused on mutants of the third helix, which provides DNA binding specificity, and charac-terized mutations that were highly associated with either ventricular (VSD) or atrioventricular (AVSD)septal defects. Individual mutants exhibited partial to complete loss of function and differences in transacti-vation capacity between the various REs. The mutants also exhibited gene dosage rather than dominanteffects on transcription. Surprisingly, all AVSD patients (22/23) had a single K183E mutation in the DNA bind-ing domain, which resulted in transcriptional inactivation. None of the VSD patients had this mutation; yet14/29 had at least one mutation in the third helix leading to either inactivation or reduction of NKX2-5transactivation. Therefore, mutations of somatic origin in the binding domains of NKX2-5 were associatedspecifically with AVSD or VSD and resulted in loss of protein function.INTRODUCTIONThe homeodomain (HD) protein Nkx2-5 is an evolutionarilyconserved transcription factor required for the organogenesisof the heart (1) and the development of the cardiac conductionsystem (2). The human NKX2-5 gene maps to chromosome5q34 and consists of two exons encoding a protein of 324amino acids. The HD portion of the protein forms threealpha helices, and helix 3 is responsible for binding specificity.Other conserved regions of Nkx2-5 are the TN regions of thetransactivation domain (TAD) and the NK2-specific domain(NK2-SD), with autoregulatory functions (3).Several transcription factors cooperate in the regulationof heart development. Results from whole animals andcellular models suggest complex functional interplays andthe necessity for proper gene dosage of these regulators(reviewed in 4–6). Deletion of both Nkx2-5 alleles in themouse results in lethality due to impaired cardiac loopingPublished by Oxford University Press 2005.

Azoarcus sp. strain BH72 is a Gram-negative proteobacterium of the beta subclass; it is a diazotrophic endophyte of graminaceous plants and can provide significant amounts of fixed nitrogen to its host plant Kallar grass. We aimed to obtain a physical map of the Azoarcus sp. strain BH72 chromosome to be directly used in functional analysis and as a part of an Azoarcus sp. BH72 genome project. A bacterial artificial chromosome (BAC) library was constructed and analysed. A representative physical map with a high density of marker genes was developed in which 64 aligned BAC clones covered almost the entire genome. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The T-box transcription factor Tbx5 is important in mammalian cardiac development. Mutations in the human TBX5 gene cause Holt-Oram syndrome (HOS), a disorder characterized by heart and upper limb deformities. To determine the role of TBX5 in non-HOS patients with complex cardiac malformations, we analyzed 68 explanted hearts from unrelated patients with various cardiac abnormalities including atrial (ASD), ventricular (VSD) and atrioventricular septal defects (AVSD). Direct sequencing detected nine mutations in diseased cardiac tissues of patients, eight of which are novel. Six mutations would affect amino acids in the T-domain, and one (c.236C>T, p.Ala79Val) is within the recently identified nuclear localization signal (NLS1) region. Further, mutations were found in patients with ASD and AVSD, but not with VSD; and mutations were absent in normal heart tissue of same patients, thus indicating somatic origin. Our results suggest a possible role of somatically occurring TBX5 mutations in congenital heart disease. We show for the first time TBX5 mutations in non-HOS associated cardiac malformations and we identified a novel missense mutation that would impact nuclear localization of TBX5. © 2004 Wiley-Liss, Inc.

NKX2-5 is a homeodomain-containing transcription factor important in cardiac development. Familial mutations in the NKX2-5 gene are associated with cardiac abnormalities, but mutations are rare in sporadic cases. We studied the pathology and molecular genetics of NKX2-5 in diseased heart tissues of 68 patients with complex congenital heart disease (CHD), particularly atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). We also studied DNA extracted from 16 normal hearts, as well as lymphocytic DNA from 50 healthy volunteers, 7 families, and 4 unrelated individuals with CHD. Direct sequencing revealed 53 NKX2-5 mutations in the diseased heart tissues, including nonsynonymous substitutions in the homeodomain of NKX2-5. We found common mutations among unrelated patients, but certain mutations were specific to VSDs and AVSDs. Many patients had multiple NKX2-5 mutations, up to 14 nonsynonymous mutations per patient in VSDs. Importantly, these nonsynonymous mutations were mainly absent in normal heart tissues of the same CHD patients, thus indicating somatic origin and mosaicism of mutations. Further, observed mutations were completely absent in normal hearts and lymphocytic DNA of healthy individuals. Our findings provide new insights for somatic NKX2-5 mutations to be of importance in congenital heart disease.

Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.

The basic helix-loop-helix (bHLH) transcription factor Hey2 (gridlock) is an important determinant of mammalian heart development, but its role in human ventricular septal defects is unknown. Hey2 functions as a repressor through the bHLH domain. By direct sequencing, we analyzed the sequences encoding the bHLH domain of the human HEY2 in 52 explanted hearts of unrelated patients with complex cardiac malformations, notably ventricular (VSD) and atrioventricular septal defects (AVSD). We found three nonsynonymous mutations, namely, c.286A>G (p.Thr96Ala), c.293A>C (p.Asp98Ala), and c.299T>C (p.Leu100Ser) affecting the second helix of HEY2 in the diseased cardiac tissues of two patients with AVSD. This result suggests a possible role of HEY2 in the regulation of ventricular septation in humans. Since the two AVSD patients carried also binding domain mutations in other cardiac-specific transcription factors, e.g. NKX2-5, TBX5, and GATA4, breakdown of combinatorial interactions of transcription factors may have contributed to the complexity of their cardiac malformations. © 2005 Wiley-Liss, Inc.

Background Parkinson's disease (PD) is a movement disorder caused by the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. The molecular basis of this neural death is unknown, but genetic predisposition and environmental factors may cause the disease. Sequence variations in N-acetyltransferase 2 (NAT2) gene leading to slow acetylation process have been associated with PD, but results are contradictory. Methods We analyzed three NAT2 genetic variations, c.481C>T, c.590G>A (p.R197Q) and c.857G>A (p.G286E), which are known to result in a slow acetylator phenotype. Using validated PCR-RFLP assays, we genotyped 243 healthy unrelated Caucasian control subjects and 124 PD patients for these genetic variations. Further, we have undertaken a systematic review of NAT2 studies on PD and we incorporated our results in a meta-analysis consisting of 10 studies, 1,206 PD patients and 1,619 control subjects. Results Overall, we did not find significant differences in polymorphic acetylation genotypes in PD and control subjects. In the meta-analysis of slow acetylators from 10 studies and representing 604/1206 PD vs. 732/1619 control subjects, a marginally significant odds ratio (OR) of 1.32 (95% CI 1.12–1.54, p < 0.05) was obtained. Re-analysis of the data to exclude the only two studies showing positive association of slow acetylators to PD, resulted in a non-significant OR (1.07, 95% CI 0.9–1.28). Furthermore, meta-analysis of studies for c.590G>A, where both allele and genotype frequencies in PD vs. control subjects were analyzed, did not give significant summary odds ratios as well. Conclusion We found little evidence for differences in polymorphic acetylation genotypes in PD and control subjects. Results of the meta-analyses did not also provide conclusive evidence for an overall association of NAT2 slow acetylator genotypes to PD.

Hypoplasia of the human heart is the most severe form of congenital heart disease (CHD) and usually lethal during early infancy. It is a leading cause of neonatal loss, especially in infants diagnosed with hypoplastic left heart syndrome (HLHS), a condition where the left side of the heart including the aorta, aortic valve, left ventricle (LV) and mitral valve are underdeveloped. The molecular causes of HLHS are unclear, but the basic helix-loop-helix (bHLH) transcription factor heart and neural crest derivatives expressed 1 (Hand1), may be a candidate culprit for this condition. The absence of Hand1 in mice resulted in the failure of rightward looping of the heart tube, a severely hypoplastic LV and outflow tract abnormalities. Nonetheless, no HAND1 mutations associated with human CHD have been reported so far. We sequenced the human HAND1 gene in heart tissues derived from 31 unrelated patients diagnosed with hypoplastic hearts. We detected in 24 of 31 hypoplastic ventricles, a common frameshift mutation (A126fs) in the bHLH domain, which is necessary for DNA binding and combinatorial interactions. The resulting mutant protein, unlike wild-type (wt) HAND1, was unable to modulate transcription of reporter constructs containing specific DNA-binding sites. Thus, in hypoplastic human hearts HAND1 function is impaired.