There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae.

AS (2016) Inversion of correia repeat enclosed elements in 'Neisseria gonorrhoeae', which will be published in final form at http://dx.doi.org/10.1099/mic.0.000394. This article may be used for non-commercial purposes in accordance with the publisher restrictions. Title:Inversion of Correia Repeat Enclosed Elements in Neisseria gonorrhoeae. Authors:Firat Elbeyioglu*, Sabrina B. Roberts*, Russell Spencer-Smith^, Madhuri Pulijala, Marta A. Zelewska, Jean-Christophe Nebel ‡ , and Lori A. S. Snyder † Affiliations:School of Life Sciences, Pharmacy, and Chemistry, Kingston University, Kingston upon Thames, UK. KT1 Short title:Invertible elements in Neisseria gonorrhoeae. Keywords:Gonococcus; phase variation; inversion; Correia Repeat Enclosed Elements; CREE Subject category:Genomics and systems biology. Word count: 2,652 Depositories:The GenBank accession numbers for the re-sequencing data of Neisseria gonorrhoeae strain NCCP11945 are SRR3547950 and (submitted SUB2025235). Abbreviations: CREE: Correia Repeat Enclosed Element Abstract:Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Within the gonococcal genome are over 100 copies of the IS-like Correia Repeat Enclosed Element, which has been predicted to be mobile within the neisserial genomes. Although there is evidence of ancestral movement of these elements, no previous study has provided evidence for current mobilisation.The Correia Repeat Enclosed Element has the ability to alter gene expression and regulation in many ways: by insertional mutagenesis; by introducing promoter elements; by generating mRNA processing sites, and by association with ncRNAs.Previous studies have compared the genomic locations of Correia Repeat Enclosed Elements in the Neisseria spp., demonstrating that otherwise identical regions have either the element or the target TA insertion site. In this study, we report for the first time movement of Correia Repeat Enclosed Elements, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from Neisseria gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample; 7 in the control sample; and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements, potentially resulting in reversible phase variable gene expression. Introduction:

Comparisons of genome sequence data between different strains and isolates of Neisseria spp., such as Neisseria gonorrhoeae, reveal that over the evolutionary history of these organisms, large scale chromosomal rearrangements have occurred. Factors within the genomes, such as repetitive sequences and prophage, are believed to have contributed to these observations. However, the timescale in which rearrangements occur is not clear, nor whether it might be expected for them to happen in the laboratory. In this study, N. gonorrhoeae was repeatedly passaged in the laboratory and assessed for large scale chromosomal rearrangements. Using gonococcal strain NCCP11945, for which there is a complete genome sequence, cultures were passaged for eight weeks in the laboratory. The resulting genomic DNA was assessed using Pulsed Field Gel Electrophoresis, comparing the results to the predicted results from the genome sequence data. Three cultures generated Pulsed Field Gel Electrophoresis patterns that varied from the genomic data and were further investigated for potential chromosomal rearrangements.

Study questionCan small genetic variants detected in the whole genome sequencing of spontaneously aborted euploid embryos give insight into possible causes of pregnancy loss?Summary answerBy filtering and prioritizing genetic variants it is possible to identify genomic variants putatively responsible for miscarriage.What is known alreadyMiscarriage is often caused to chromosomal aneuploidies of the gametes but it can also have other genetic causes like small mutations, both de novo or inherited from parents. The analysis of genomic sequences of miscarried embryos has mostly focused on rare variation, and been carried out using criteria and methods that are difficult to reproduce. The role of small mutations has been scantily investigated so far.Study design, size, durationThis is a monocentric observational study. The study includes the data analysis of 46 embryos obtained from women experiencing pregnancy loss recruited by the University of Ferrara from 2017 to 2018. The study was approved by the Ethical committee of Emilia-Romagna (CE/FE 170475).Participants/materials, setting, methodsThe participants are forty-six women, mostly European (87%) diagnosed with first (n=25, av.age 32.7) or recurrent (n=21, av.age 36.5) miscarriage. Embryonic DNA was prepared form chorionic villi and used to select euploid embryos using quantitative PCR, comparative genomic hybridiztion and shallow sequencing of random genomic regions. Euploid embryos were whole-genome sequenced at 30X using Illumina short-reads technology and genomic sequences were used to identify genetic variants. Variants were annotated integrating information from Ensembl100 and literature knowledge on genes associated with embryonic development, miscarriages, lethality, cell cycle. Following annotation, variants were filtered to prioritize putatively detrimental variants in genes that are relevant for embryonic development using a pipeline that we developed. The code is available on gitHub (ezcn/grep).Main results and the role of chanceOur pipeline prioritized 439 putatively causative single nucleotide polymorphisms among 11M variants discovered in ten embryos. By systematic investigation of all coding regions, 47 genes per embryo were selected. Among them STAG2, known in literature for its role in congenital and developmental disorders as well as in cancer, TLE4 a key gene in embryonic development, expressed in both embryonic and extraembryonic tissues in the Wnt and Notch signalling pathways, and FMNL2, involved in cell motility with a major role in driving cell migration. Our analysis is fully reproducible (our code is open-source), and we take measures to increase its robustness to false positives by excluding genes with >5% chance to be selected in a control population.Limitations, reasons for cautionThis pilot study has major limitations in sample size and lack of integration of the parental genomic information. Despite being encouraging, the results need to be interpreted with caution as functional analyses are required to validate the hypotheses that have been generated. Although we have developed a robust and scalable methodology for prioritizing genetic variants, we have not yet extended it beyond the coding regions of the genome.Wider implications of the findingsThis pilot study demonstrate that analysis of genome sequencing can help to clarify the causes of idiopathic miscarriages and provides initial results from the analysis of ten euploid embryos, discovering plausible candidate genes and variants. This study provides guidance for a larger study. Results of this and following wider studies can be used to test genetic predisposition to miscarriages in parents that are planning to conceive or undergoing preimplantation genetic testing. In a wider context, the results of this study might be relevant for genetic counseling and risk management in miscarriagesStudy funding/competing interest(s)A.C. is a full time employee of Igenomix. A.D.M. was employee of Igenomix while working on this project. I.D.B., P.D.A., G.E., S.D.B. are full time employees of the MeriGen Research. All other authors declare that they have no conflicts of interest.

Miscarriage is the spontaneous termination of a pregnancy before 24 weeks of gestation. We studied the genome of euploid miscarried embryos from mothers in the range of healthy adult individuals to understand genetic susceptibility to miscarriage not caused by chromosomal aneuploidies. We developed gp , a pipeline that we used to prioritize 439 unique variants in 399 genes, including genes known to be associated with miscarriages. Among the prioritized genes we found STAG2 coding for the cohesin complex subunit, for which inactivation in mouse is lethal, and TLE4 a target of Notch and Wnt, physically interacting with a region on chromosome 9 associated to miscarriages.