Increased Gene Dosage Research Articles

Helicase/SUMO-targeted ubiquitin ligase Uls1 interacts with the Holliday junction resolvase Yen1.

Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures.

Over 2000-Fold Increased Production of the Leaderless Bacteriocin Garvicin KS by Increasing Gene Dose and Optimization of Culture Conditions.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 is, however, relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes, a new medium formulation was made that increased GarKS production about 60-fold compared to that achieved in GM17. The new medium was composed of pasteurized milk and tryptone (PM-T). GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing the gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and cultivation at a constant pH 6 and a 50–60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.

DNA Ligase IV Prevents Replication Fork Stalling and Promotes Cellular Proliferation in Triple Negative Breast Cancer.

DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.

Epigenetics, aging, and glycolysis

Aging Aging brings reduced fitness and increased incidence of disease and death. Epigenetic changes are thought to be associated with various aging processes. By using CRISPR-Cas9 mutagenesis in the model fly, Drosophila , Ma et al. found that aging was associated with a loss of fidelity in histone modifications mediated by Polycomb proteins—specifically, a reduction in the repressive epigenetic mark called H3K27me2/3. If the Polycomb protein PRC2 was mutated, H3K27me2/3 was lost, glycolysis was elevated, and life span was restored. Transgenically increasing gene dosage for glycolytic enzymes also promoted life span, locomotion, and resistance to oxidative stress. eLife 10.7554/eLife.35368 (2018).

Abstract 1482: SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL)

Abstract Individuals with Down syndrome (DS) are highly predisposed to develop hematopoietic malignancies, especially acute megakaryoblastic leukemia (DS-AMKL). Generally, genes located on chromosome 21 tend to be increased in Down syndrome patients, but how these individual genes contribute to development of DS-AMKL is still unclear. SON, a gene located on chromosome 21, is upregulated in Down syndrome patients, and it has been reported that DS-AMKL cells have higher level of SON expression. The SON protein functions as a splicing factor and a transcription regulator in leukemia but the role of SON upregulation in DS-AMKL pathogenesis is still not fully elucidated. In addition, runt-related transcription factor 1 (RUNX1) is also located on chromosome 21, but its expression level is downregulated in DS-AMKL patients despite the increased gene dosage. We show that SON represses RUNX1 expression and blocks terminal megakaryocytic differentiation in DS-AMKL. Knockdown of SON in DS-AMKL cell lines leads to morphologic changes and increased cell adhesion. Moreover, cell surface marker analyses demonstrate that the depletion of SON induces increased CD41+ CD61+ populations as well as polyploidy cells, indicating megakaryocytic differentiation. Consistent with these results, SON deletion causes upregulation of RUNX1, which is a critical factor in the megakaryocytic differentiation. Furthermore, our data show that knockdown of SON increases several megakaryocytic transcription factors and decreases erythropoietic transcription factors, facilitating terminal megakaryocytic differentiation of leukemic blasts. Mechanistically, SON binds to the proximal promoter region as well as two intronic enhancer regions upstream of the proximal promoter at the RUNX1 gene locus, and suppresses transcription of RUNX1 by lowering the level of H3K4me3. Taken together, we propose that the increased genomic dosage of SON in Down syndrome impairs terminal differentiation of megakaryo-erythroblasts into mature megakaryocytes through binding to the RUNX1 promoter/enhancer to repress its transcription, which contributes to the higher incidence of AMKL in Down syndrome patients. Our results reveal a novel connection of SON to DS-AMKL pathogenesis and provide clinical importance of SON upregulation in DS. These findings suggest that SON could be a promising therapeutic target for DS-AMKL patients. Citation Format: Lana Vukadin, Eun young Park, Jung Hyun Kim, Erin Eun Young Ahn. SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1482.

Abstract 391: Evolutionary trajectories and KRAS gene dosage define pancreatic cancer phenotypes

Abstract Pancreatic ductal adenocarcinoma (PDAC) has frequent alterations in few genes (KRAS, CDKN2A/TP53/SMAD4) and extensive heterogeneity of cancer drivers beyond. The expectation that mutational landscapes of rare drivers could explain phenotypic diversity has -with few exceptions- not come true. Likewise, PDAC metastasis is not understood, and comparisons of primary/metastasis pairs did not find recurrently mutated “metastasis genes”. Here we show that key aspects of PDAC biology are defined by gene-dosage variation of PDAC signature genes, evolving along distinct evolutionary routes. We found increased gene dosage of the initiating KRAS mutation (KRASMUT-iGD) in human PDAC precursors. Mouse models revealed the importance of KrasMUT-iGD for both, early progression and metastasis, rationalizing the high frequency of PDAC dissemination at diagnosis. To overcome limitations posed to gene dosage studies by PDAC´s stroma-richness, we developed murine cell culture resources comprising 135 primaries/metastases. Integrative analyses of their genomes, transcriptomes and tumor phenotypes, combined with human studies and functional analyses revealed a series of additional KrasMUT-dosage effects: different KrasMUT-levels define distinct cellular morphologies, histopathologies and clinical outcomes, with highest KrasMUT-expression underlying the most aggressive undifferentiated phenotypes. We also observed KrasMUT-dosage-associated cellular plasticity, including epithelial-to-mesenchymal transition. Mechanistically, oncogenic dosage-variation is linked to distinct evolutionary routes, characterized by defined types/states of tumor-suppressor alterations: Phylogenetic tracking studies revealed convergent evolution of KrasMUT-iGD-gains, with dependence on prior homozygous Cdkn2a- or Trp53-loss. By contrast, in Cdkn2aHET cancers, amplifications of known and novel oncogenes (Myc, Yap1, Nfkb2) collaborate with KrasMUT-HET to drive progression, yet with lower metastatic potential. These results also reveal oncogene-selective/context-dependent Cdkn2a-haploinsufficiency, for which Tgfβ pathway alterations provide permissiveness. Our study uncovers universal principles underlying PDAC biology and phenotypic diversification. It describes evolutionary trajectories, identifies their genetic hallmarks and shows how oncogenic dosage-variation is differentially licensed along individual routes to control critical disease characteristics, including early progression, histopathology, metastasis, cellular plasticity and clinical aggressiveness. Citation Format: Sebastian Mueller, Thomas Engleitner, Roman Maresch, Magdalena Zukowska, Sebastian Lange, Thorsten Kaltenbacher, Björn Konukiewitz, Rupert Öllinger, Maximilian Zwiebel, Alex Strong, Hsi-Yu Yen, Ruby Banerjee, Sandra Louzada, Beiyuan Fu, Barbara Seidler, Juliana Götzfried, Kathleen Schuck, Zonera Hassan, Nina Schönhuber, Sabine Klein, Christian Veltkamp, Mathias Friedrich, Lena Rad, Maxim Barenboim, Christoph Ziegenhain, Julia Hess, Oliver M. Dovey, Stefan Eser, Swati Parekh, Fernando Constantino-Casas, Jorge de la Rosa, Marta I. Sierra, Mario Fraga, Julia Mayerle, Günter Klöppel, Roland M. Schmid, Juan Cadiñanos, Pentao Liu, George Vassiliou, Wilko Weichert, Katja Steiger, Wolfgang Enard, Fengtang Yang, Kristian Unger, Günter Schneider, Ignacio Varela, Allan Bradley, Dieter Saur, Roland Rad. Evolutionary trajectories and KRAS gene dosage define pancreatic cancer phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 391.

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments.

Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

Tuning Vector Stability and Integration Frequency Elevates Functional GPCR Production and Homogeneity in Saccharomyces cerevisiae.

Membrane proteins play a valuable role in biotechnology, yet the difficulty of producing high yields of functional membrane protein limits their use in synthetic biology. The practical application of G protein-coupled receptors in whole cell biosensors, for example, is restricted to those that are functionally produced at the cell surface in the chosen host, limiting the range of detectable molecules. Here, we present a facile approach to significantly improve the yield and homogeneity of functional membrane proteins in Saccharomyces cerevisiae by altering only the choice of expression vector. Expression of a model GPCR, the human adenosine A2a receptor, from commonly used centromeric and episomal vectors leads to low yields and cellular heterogeneity due to plasmid loss in 20-90% of the cell population. In contrast, homogeneous production of GPCR is attained using a multisite integrating vector or a novel, modified high copy vector that does not require genomic integration or addition of any selection agents. Finally, we introduce a FACS-based screen, which enables rapid isolation of cells with 4- to 15-fold increases in gene dosage and up to a 9-fold increase in functional protein yield without loss of homogeneity compared to a strain isolated through conventional, low-throughput methods. These results can be extended to improve the cellular homogeneity and yield of other membrane proteins, expanding the repertoire of useful receptors for synthetic biology applications.

Precision medicine approaches may be the future for CRLF2 rearranged Down Syndrome Acute Lymphoblastic Leukaemia patients

Breakthrough studies over the past decade have uncovered unique gene fusions implicated in acute lymphoblastic leukaemia (ALL). The critical gene, cytokine receptor-like factor 2 (CRLF2), is rearranged in 5–16% of B-ALL, comprising 50% of Philadelphia-like ALL and cooperates with genomic lesions in the Jak, Mapk and Ras signalling pathways. Children with Down Syndrome (DS) have a predisposition to developing CRLF2 rearranged-ALL which is observed in 60% of DS-ALL patients. These patients experience a poor survival outcome. Mutations of genes involved in epigenetic regulation are more prevalent in DS-ALL patients than non-DS ALL patients, highlighting the potential for alternative treatment strategies. DS-ALL patients also suffer greater treatment-related toxicity from current ALL treatment regimens compared to non-DS-ALL patients. An increased gene dosage of critical genes on chromosome 21 which have roles in purine synthesis and folate transport may contribute. As the genomic landscape of DS-ALL patients is different to non-DS-ALL patients, targeted therapies for individual lesions may improve outcomes. Therapeutically targeting each rearrangement with targeted or combination therapy that will perturb the transforming signalling pathways will likely improve the poor survival rates of this subset of patients.

Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage.

Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, crtS, on Chr1. The mechanism by which replication of crtS allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra crtS copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated crtS copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that crtS activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.

Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity.

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies.

Life-stage-associated remodelling of lipid metabolism regulation in Atlantic salmon.

Atlantic salmon migrates from rivers to sea to feed, grow and develop gonads before returning to spawn in freshwater. The transition to marine habitats is associated with dramatic changes in the environment, including water salinity, exposure to pathogens and shift in dietary lipid availability. Many changes in physiology and metabolism occur across this life-stage transition, but little is known about the molecular nature of these changes. Here, we use a long-term feeding experiment to study transcriptional regulation of lipid metabolism in Atlantic salmon gut and liver in both fresh- and saltwater. We find that lipid metabolism becomes significantly less plastic to differences in dietary lipid composition when salmon transitions to saltwater and experiences increased dietary lipid availability. Expression of genes in liver relating to lipogenesis and lipid transport decreases overall and becomes less responsive to diet, while genes for lipid uptake in gut become more highly expressed. Finally, analyses of evolutionary consequences of the salmonid-specific whole-genome duplication on lipid metabolism reveal several pathways with significantly different (p<.05) duplicate retention or duplicate regulatory conservation. We also find a limited number of cases where the whole-genome duplication has resulted in an increased gene dosage. In conclusion, we find variable and pathway-specific effects of the salmonid genome duplication on lipid metabolism genes. A clear life-stage-associated shift in lipid metabolism regulation is evident, and we hypothesize this to be, at least partly, driven by nondietary factors such as the preparatory remodelling of gene regulation and physiology prior to sea migration.

Benzene induced resistance in exposed Drosophila melanogaster: Outcome of improved detoxification and gene modulation

Adaptive behaviour of an organism has relevance towards developing better resistance in subsequent generations following xenobiotic exposures. Using a genetically tractable and functional insect model, Drosophila melanogaster, we aimed to examine the resistance of the organism against repeated exposures of benzene, an industrial and environmental-chemical and a class I human carcinogen. While 100 mM benzene exposure to one-day old flies for seven days caused ∼95% mortality (F0), its exposure to subsequent generations of flies led a significant decrease in mortality with maximum survival (∼85%) as evident at F28 generation. While burden of benzene and its toxic metabolites was higher in initial generations, in latter generations (F24-F28), concentrations of less toxic metabolites were higher. In parallel, improved metabolism, less oxidative stress, less induction of hsp60 and hsp70 and higher induction of hsp26 and hsp27 along with increased gene dose ratio of three genes (cyp6g1, mrp1, and cyp12d1) were observed in latter generations of benzene exposed flies with maximum benefit accrued in F28 generation. The resistance developed in flies of F28 generation had a negative impact on reproduction which might be due to a cost against selection. The study demonstrates development of benzene resistance in Drosophila with permanent genetic changes.

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

The poor correlation of mutational landscapes with phenotypes limits our understanding of pancreatic ductal adenocarcinoma (PDAC) pathogenesis and metastasis. Here we show a critical role of oncogenic dosage-variation in PDAC biology and phenotypic diversification. We found gene-dosage increase of mutant KRASMUT in human PDAC precursors, driving both early tumorigenesis and metastasis, thus rationalizing early PDAC dissemination. To overcome limitations posed to gene-dosage studies by PDAC´s stroma-richness we developed large cell culture resources of metastatic mouse PDAC. Integration of their genomes, transcriptomes and tumor phenotypes with functional studies and human data, revealed additional widespread effects of oncogenic dosage-variation on cell morphology/plasticity, histopathology and clinical outcome, with highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, yet with lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumor-suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive early progression and shape downstream PDAC biology. Our study uncovers universal principles in Ras-driven oncogenesis with potential relevance beyond pancreatic cancer.

Computational Characterization of the Promoter Region of SMN2 (Survival of Motor Neuron) Gene

SMN (Survival of Motor Neuron) has two isotypes; SMN1 and SMN2 encoding for FL-SMN protein (functional SMN) and ?7SMN protein (non functional). The lack of SMN1 produce spinal muscular atrophy (SMA) and the increasing gene dosage of SMN2 have been shown to decrease the severity of the SMA. The core promoter region of the SMN genes is still not identified but previous literature have shown these genes to be regulated by a 4.6 kb region up stream of the transcription start site (TSS). There was a need of computational analysis of this region as the presence of important features can help to develop a strategy for gene therapy against spinal muscular atrophy (SMA). In this analysis, the open reading frames, Pribnow box sequences, restriction sites and the transcriptional factor binding sites were identified. Important restriction sites were determined with in 15 open reading frames. Total of 15 ORFs and 24 nested ORFs were determined with 7 and 11 ORFs on the complementary strand. These ORFs contained 15 TATA box sequences reflecting the diverse function integrity of SMN promoter region. The whole data was used for the prediction of the promoter region of the SMN. Up to the best of our knowledge this is the first reported study in the literature reflecting the computational analysis of the ~4.6 kb expected promoter region of the SMN genes towards analyzing the core promoter region of the SMN genes in the future studies.

The presence of PD-1 positive tumor infiltrating lymphocytes in triple negative breast cancers is associated with a favorable outcome of disease.

Triple negative breast cancer patients have a poor course of disease not least because of limited treatment options however immunotherapy by targeting the PD-1/PD-L1 checkpoint system is a promising strategy to improve the outcome. Here we systematically investigated the expression of PD-1 on tumor infiltrating lymphocytes and PD-L1 on both tumor and infiltrated immune cells. Moreover, the PD-L1 gene status in tumor cells was assessed.103 tissue microarray samples derived from triple negative breast cancer specimens were immunohistochemically stained against PD-1 and PD-L1. Dual marker fluorescence in-situ hybridization was applied to the PD-L1 gene and centromere region of chromosome 9. The disease free and overall survival rates were determined as a function of the PD-1/PD-L1 status.A slight gain of the PD-L1 gene region was found in 55% of all samples but an elevated PD-L1/cen9 ratio was rather rare (7%). An increased gene dose is not associated with an enhanced protein expression and the PD-L1 expression only weakly correlates with the amount of immune cell infiltration. Instead, we found an association of PD-L1 expression on tumor and immune cells, respectively. Notably, the PD-1 expression on immune cells is associated with a favorable disease free and overall survival. PD-1 expression indicates an enhanced immunological anti-tumor activity and represents a favorable prognostic impact. A deeper understanding of factors that affect the regulation and function of the PD-1/PD-L1 system is required to establish predictive variables and to utilize the system for therapeutic intervention of triple negative breast cancer patients.

HUWE1 variants cause dominant X-linked intellectual disability: a clinical study of 21 patients

Whole-gene duplications and missense variants in the HUWE1 gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1 variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G > A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1 missense variants may cause syndromic ID in both males and females.

Searching for new pharmacological targets for the treatment of Alzheimer's disease in Down syndrome

Individuals with Down syndrome are at increased risk of developing Alzheimer's disease due to increase gene dosage resulting from chromosome 21 triplication. Although virtually all adults with Down syndrome will exhibit the major neuropathological hallmarks that define Alzheimer's disease, not all of them will develop the clinical symptoms associated with this disorder (i.e. dementia). Therefore, a good understanding of the pathophysiology of Alzheimer's disease in Down syndrome will be crucial for the identification of novel pharmacological targets to develop disease-modifying therapies for the benefit of Down syndrome individuals and for Alzheimer's sufferers alike. The study of biomarkers will also be essential for the development of better screening tools to identify dementia at its incipient stages. This review discusses the best-validated pharmacological targets for the treatment of cognitive impairment and Alzheimer's disease in Down syndrome. We further examine the relevance of newly discovered biological markers for earlier dementia diagnosis in this population.

Vasopressin deletion is associated with sex-specific shifts in the gut microbiome

ABSTRACTBrattleboro rats harbor a spontaneous deletion of the arginine-vasopressin (Avp) gene. In addition to diabetes insipidus, these rats exhibit low levels of anxiety and depressive behaviors. Recent work on the gut-brain axis has revealed that gut microbiota can influence anxiety behaviors. Therefore, we studied the effects of Avp gene deletion on gut microbiota. Since Avp gene expression is sexually different, we also examined how Avp deletion affects sex differences in gut microbiota. Males and females show modest but differentiated shifts in taxa abundance across 3 separate Avp deletion genotypes: wildtype (WT), heterozygous (Het) and AVP-deficient Brattleboro (KO) rats. For each sex, we found examples of taxa that have been shown to modulate anxiety behavior, in a manner that correlates with anxiety behavior observed in homozygous knockout Brattleboro rats. One prominent example is Lactobacillus, which has been reported to be anxiolytic: Lactobacillus was found to increase in abundance in inverse proportion to increasing gene dosage (most abundant in KO rats). This genotype effect of Lactobacillus abundance was not found when females were analyzed independently. Therefore, Avp deletion appears to affect microbiota composition in a sexually differentiated manner.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 16

ObjectiveWe present prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 16. Case reportA 28-year-old woman underwent amniocentesis at 17 weeks of gestation because of abnormal maternal serum screening for Down syndrome. Amniocentesis revealed a karyotype of 47,XY,+mar[5]/46,XY[9]. Parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. Array comparative genomic hybridization (aCGH) analysis of cultured amniocytes revealed a de novo 16% gene dosage increase of 16q11.2-q22.1. Repeat amniocentesis at 21 weeks of gestation revealed a karyotype of 47,XY,+mar[10]/46,XY[31]. aCGH analysis of uncultured amniocytes revealed a result of arr 16q11.2q22.1 (46,492,626–68,867,969) × 2.20 with a log2 ratio of 0.15 encompassing RPGRIP1L, FTO, SLC6A2, BBS2 and CDH1. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes detected partial trisomy 16q in 36/137 (26.3%) of uncultured amniocytes. Polymorphic DNA marker analysis on amniocytes and parental bloods excluded uniparental disomy 16. Premature labor occurred at 25 weeks of gestation, and a 585-g male baby without craniofacial dysmorphism was delivered and survived. At age 1½ years, pediatric follow-ups revealed normal psychomotor development, normal body weight, short stature, congenital hypothyroidism, hearing impairment and hypospadias in the neonate, and the peripheral blood had a karyotype of 46,XY in 40 cultured lymphocytes. ConclusionaCGH, interphase FISH and polymorphic DNA marker analyses of uncultured amniocytes are useful for confirmation of prenatally detected mosaic sSMCs at amniocentesis.