Cdx1 and Cdx2 have Overlapping Functions in Anteroposterior Patterning and Posterior Axis Elongation

Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogues, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. We have analysed the genetic interactions between Cdx1 and Cdx2 in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. We conclude that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminding of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice

Correction: Association of Rare Genetic Variants in Opioid Receptors with Tourette Syndrome.

[This corrects the article DOI: 10.5334/tohm.464.]

Association of Rare Genetic Variants in Opioid Receptors with Tourette Syndrome

Background: Genes involved in Tourette syndrome (TS) remain largely unknown. We aimed to identify genetic factors contributing to TS in a French cohort of 120 individuals using a combination of hypothesis-driven and exome-sequencing approaches. Methods: We first sequenced exons of SLITRK1-6 and HDC in the TS cohort and subsequently sequenced the exome of 12 individuals harboring rare variants in these genes to find additional rare variants contributing to the disorder under the hypothesis of oligogenic inheritance. We further screened three candidate genes (OPRK1, PCDH10, and NTSR2) preferentially expressed in the basal ganglia, and three additional genes involved in neurotensin and opioid signaling (OPRM1, NTS, and NTSR1), and compared variant frequencies in TS patients and 788 matched control individuals. We also investigated the impact of altering the expression of Oprk1 in zebrafish. Results: Thirteen ultrarare missense variants of SLITRK1-6 and HDC were identified in 12 patients. Exome sequencing in these patients revealed rare possibly deleterious variants in 3,041 genes, 54 of which were preferentially expressed in the basal ganglia. Comparison of variant frequencies altering selected candidate genes in TS and control individuals revealed an excess of potentially disrupting variants in OPRK1, encoding the opioid kappa receptor, in TS patients. Accordingly, we show that downregulation of the Oprk1 orthologue in zebrafish induces a hyperkinetic phenotype in early development. Discussion: These results support a heterogeneous and complex genetic etiology of TS, possibly involving rare variants altering the opioid pathway in some individuals, which could represent a novel therapeutic target in this disorder

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BACKGROUND: SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. METHODS: Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. RESULTS: We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p\u2009<\u20090.005), an increased number of cells in the G0/G1 phase (p\u2009<\u20090.001), and an increased mortality because of apoptosis (p\u2009<\u20090.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p\u2009<\u20090.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways. CONCLUSIONS: Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge. \ua9 2013 Mancini et al.; licensee BioMed Central Ltd

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Intronic ATTTC repeat expansions in STARD7 in familial adult myoclonic epilepsy linked to chromosome 2

Familial Adult Myoclonic Epilepsy (FAME) is characterised by cortical myoclonic tremor usually from the second decade of life and overt myoclonic or generalised tonic-clonic seizures. Four independent loci have been implicated in FAME on chromosomes (chr) 2, 3, 5 and 8. Using whole genome sequencing and repeat primed PCR, we provide evidence that chr2-linked FAME (FAME2) is caused by an expansion of an ATTTC pentamer within the first intron of STARD7. The ATTTC expansions segregate in 158/158 individuals typically affected by FAME from 22 pedigrees including 16 previously reported families recruited worldwide. RNA sequencing from patient derived fibroblasts shows no accumulation of the AUUUU or AUUUC repeat sequences and STARD7 gene expression is not affected. These data, in combination with other genes bearing similar mutations that have been implicated in FAME, suggest ATTTC expansions may cause this disorder, irrespective of the genomic locus involvedSupplementary Information: Supplementary Data 1; Supplementary Data 2; Reporting Summary.NHMRC; Women’s and Children’s Hospital Research Foundation; Muir Maxwell Trust; Epilepsy Society; The European Fund for Regional Development; The province of Friesland, Dystonia Medical Research Foundation; Stichting Wetenschapsfonds Dystonie Vereniging; Fonds Psychische Gezondheid; Phelps Stichting; The Italian Ministry of Health; Istituto Superiore di Sanità, Italy; Undiagnosed Disease Network Italy; The Fondation maladies rares, University Hospital Essen and UK Department of Health’s NIHR.https://www.nature.com/ncommspm2020Neurolog

Repression of embryonic genome: how, why?

L’expression du génome de l’embryon a été étudiée en utilisant des lapins dont la lignée germinale a été enrichie avec un gène marqueur associé au promoteur du gène tk du virus de l’herpès, et à des séquences régula trices dérivées d’introns et d’exons du gène humain hprtet de la séquence F101 du polyome. Cette construction, nommée UPytl2LacZ, s’exprime dans les cellules de l’embryon transgénique collecté au stade une cellule et maintenu en culture. Lorsqu’il est d’origine paternelle, le transgène ne s’exprime in vitro qu’à partir du stade 8 cellules alors que d’origine maternelle il s’exprime à partir du stade 16 cellules. Ainsi, in vitro, l’expression du génome est réprimée pendant les premiers stades de développement de l’embryon de lapin. La répression du génome d’origine maternelle a une durée plus longue que celle du génome d’origine paternelle ; la différence correspond au temps nécessaire à la réalisation un cycle cellulaire supplémentaire. La présence d’aphidicoline, un inhibiteur de synthèse de l’ADN, dès le début de la culture s’oppose au passage de l’embryon du stade 2 au stade 4 cellules. Dans ces conditions, le transgène n’est plus exprimé quelle que soit l’origine germinale du gène. L’addition plus tardive de l’aphidicoline dans le milieu de culture, qui permet d’arrêter le développement de l’embryon au stade 4 cellules, réduit encore significativement l’expression du génome d’origine paternelle ou maternelle. Ces résultats montrent que la transcription devient possible seulement après la réalisation des phases de synthèse de l’ADN, ce qui suggère qu’une réoganisation fonctionnelle du génome de l’embryon est nécessaire pour la levée de la répression de la transcription

Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication.

International audienceIn the mouse, transcriptional permissiveness is established in the fertilized egg prior to the activation of zygotic genes at the 2-cell stage. Therefore, gene inactivity initiated at the end of gametogenesis results from a complex process, involving more than an inhibition of the basal transcriptional apparatus. We have examined the ability of the first intron (I1) of the human hypoxanthine phosphoribosyl transferase gene, which functions as an enhancer in embryonic stem cells, to activate a reporter gene when placed proximally to or at a distance from the HSV-tk promoter, or when integrated into the mouse genome as part of a stable transgene. In microinjected embryos, I1 functions as an enhancer sequence; however, its competence for long-range activation appears only after the late 1-cell stage and depends on the first DNA replication. Moreover, activation of microinjected transgenes from proximal enhancers occurs in the late 2-cell embryo and in the male pronucleus of 1-cell embryos blocked for DNA replication; whereas, for integrated transgenes, proximal enhancer activity is subject to position effects in the 2-cell embryo and first occurs at the 2- or 4-cell stage, but only after completion of DNA replication. Therefore, the absence of long-range activation and a non-permissive genomic state (the relief of which both depend on DNA replication), together with an inactive transcriptional apparatus, appear to converge to prevent any gene activity in the 1-cell embryo. We propose that the embryo exploits the process of DNA replication to relieve the transcriptionally repressive state that was initially established to fulfil two purposes: (1) to arrest maternal gene expression in the maturing oocyte and (2) to protect the unicellular egg and 1-cell embryo from premature differentiation. Reactivation of gene expression by DNA replication would therefore serve to coordinate cell proliferation and differentiation in the preimplantation embryo

CpG content affects gene silencing in mice: evidence from novel transgenes

International audienceBackgroundTransgenes are often engineered using regulatory elements from distantly related genomes. Although correct expression patterns are frequently achieved even in transgenic mice, inappropriate expression, especially with promoters of widely expressed genes, has been reported. DNA methylation has been implicated in the aberrant expression, but the mechanism by which the methylation of a CpG-rich sequence can perturb the functioning of a promoter is unknown.ResultsWe describe a novel method for analyzing epigenetic controls that allows direct testing of CpGs involvement by using LacZ reporter genes with a CpG content varying from high to zero that are combined with a CpG island-containing promoter of a widely expressed gene - the α-subunit of the translation elongation factor 1. Our data revealed that a LacZ transgene with null CpG content abolished the strong transgene repression observed in the somatic tissues of transgenic lines with higher CpG content. Investigation of transgene expression and methylation patterns suggests that during de novo methylation of the genome the CpG island-containing promoter escapes methylation only when combined with the CpG-null transgene. In the other transgenic lines, methylation of the promoter may have led to transcriptional silencing.ConclusionsWe demonstrate that the density of CpG sequences in the transcribed regions of transgenes can have a causal role in repression of transcription. These results show that the mechanism by which CpG islands escape de novo methylation is sensitive to CpG density of adjacent sequences. These findings are of importance for the design of transgenes for controlled expression

Establishment and relief of CpG-dependent transgene repression during germ line passage and mouse development

Methylation of genomic DNA at CpG sequences has a repressive effect on gene expression in vertebrates. The level of methylation of the genome varies widely during development in mammals. The DNA of early germ cells and blastocysts is largely hypomethylated but that of implantation embryos is hypermethylated. To test whether these variations might affect gene expression, we have studied the pattern of expression of a CpG-rich and of a CpG-poor LacZ transgene driven by a strong promotor of an ubiquitous gene. We find that the changes of the expression of the CpG-rich LacZ gene directly correlate with variations of methylation and that lowering the level of CpG in the reporter gene decreases dramatically its susceptibility to these variations. Therefore, in association with fluctuations in genome methylation a CpG-dependent system of repression must control positively and negatively gene expression during development and gametogenesis. We also find that the repression of the CpG-rich LacZ reporter can be abolished in 2-cell embryo by inhibitors of histone deacetylases suggesting that, at least at this stage, this CpG-dependent repression acts through histone deacetylation. In addition, we show that the β-globin locus control region and other genomic elements completely reverse the repression established at implantation in embryonic cells. The complex patterns of expression of the CpG-rich and poor LacZ reporter genes during gametogenesis and development-including a sex-dependent expression in the zygotic nucleus before the morula stage-are described. The possible implication of the constraints imposed on gene expression by DNA methylation revealed by this study are discussed