Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers.

Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored

Analysis of chromosome condensation in saccharomyces.

Eukaryotic chromosomes reach their stable rod-shaped appearance in mitosis in a reaction dependent on the evolutionarily conserved condensin complex. It is currently unknown how and where condensin associates with chromosomes. Here, we analyse condensin binding to budding yeast chromosomes by immunoprecipitation followed by hybridization on high resolution oligonucleotide tiling arrays. We observe that condensin binding sites coincide with those of the RNA polymerase III transcription factor TFIIIC and the loading factor Scc2/4 of the related cohesin complex. Both TOc and Scc2/4 facilitate condensin loading onto chromosomes. An isolated ectopic B box motif is sufficient to prime formation of a condensin binding site. While cohesin translocates away from these loading sites, condensin persists there. This defines the loading sites of cohesin and condensin and explains how an alternating pattern of these complexes along chromosomes is established. The findings have important implications for the fields of chromosome segregegation and nuclear structure in interphase and mitosis. The identification of SMC loading sites will allow targeted probing of eukaryotic chromosomes. Beside condensation, mitotic chromosome segregation also depends on the chromosomal condensin complex. Without condensin, sister chromatids fail to resolve causing anaphase bridges and chromosome breakage. How condensin promotes sister chromatid resolution is unknown. We have used the budding yeast rDNA as a model locus, whose segregation depends on condensin activity during anaphase. We show that anaphase bridges in a condensin mutant are resolved by ectopic expression of a foreign (Chlorella vims) but not endogenous yeast topoisomerase II (topo II). This suggests that catenation prevents sister rDNA segregation, and that yeast topo II is ineffective in decatenating the rDNA in the absence of condensin. We furthermore find that expression of Chlorella virus topo II in wild type cells advances the normally late segregation timing of the rDNA locus. This suggests catenation is a mean for the cell to provide rDNA cohesion up to late anaphase, when condensin promotes decatenation. This provides the first direct evidence of condensin's role in the disengagement of topologically connected sister chromatids

Methods development for the investigation of the mammalian genome radial architecture : the quantitative side

The nucleus of mammalian cells cradles the genome, an ensemble of nucleic acid macromolecular polymers that store information in a physical form. For a cell to perform life-sustaining processes, reading and utilizing the information encoded in the genome monomers’ sequence is necessary. Considerable attention has been paid to these processes since their discovery, leading to remarkable breakthroughs in our understanding of basic cell biology and the Genetics field’s birth. In the past two decades, the focus has shifted from this one-dimensional approach to a more spatio-temporal perspective. It is now clear that the genome has a complex architecture, with a multitude of organizational levels at different scales. Additionally, genome architecture interplays with gene expression, and alterations to its spatial organization associate with various pathologies like cancer, premature-aging diseases, and male infertility. In this thesis, we present the development of two methods enabling the investigation of genome architecture. In Paper I, we established iFISH, a full-stack workflow for easy DNA fluorescence in situ hybridization (FISH) setup and application. Specifically, iFISH includes a novel and accurately crafted database of 40 nt long oligonucleotide sequences for labeling specific human genomic loci. iFISH 40-mers provide a strikingly higher genomic coverage and shorter interoligo distance than other state-of-the-art databases. Moreover, the iFISH database of homologous sequences allows for the design of a 96-oligo probe in more than half of the ten kb-wide genomic regions and more than 85% of 15 kb-wide genomic regions (against a 15-30% for other databases). iFISH also includes a computational tool, easily accessible and usable via a web-based graphical user interface, for the automatic selection of optimal sets of oligos (i.e., probe design), for single-probe or homogeneous multi-probe (i.e., spotting) labeling. We applied our computational pipeline to design a total of 330 DNA FISH probes, covering all human chromosomes homogeneously, with an inter-probe distance of 10 Mb for chromosomes 1 to 16 and X and of 5 Mb for chromosomes 17 to 22. Additionally, we systematically and individually tested most probes, whose sequences are readily available for the community to download and utilize. Furthermore, we built upon cutting-edge sequence amplification methods to provide an inexpensive and straightforward protocol for the large-scale amplification of DNA FISH probes starting from relatively low concentrated oligopools. To this end, we designed a set of novel 20-mer sequences orthogonal to the human genome and compatible with the probe-specific PCR steps of the amplification protocol. Finally, we showcased the extensive applicability and flexibility of the iFISH workflow in human IMR90 fibroblast cells, revealing the importance of a dense label sampling for correct chromatin volume estimation, and in human embryonic stem cells, uncovering overall less distinct chromosome territories, and a remarkable lack of chromosome territoriality in a subset of cells. Altogether, these results support iFISH as an empowering set of tools and resources for the research community, freely accessible online at https://www.ifish4u.org. In Paper II, we presented Genomic loci Positioning by sequencing (a.k.a., GPSeq), a method for the genome-wide measurement of genomic loci position along the nuclear radius. GPSeq follows a straightforward protocol based on a simple and elegant concept: nuclear diffusion proceeds from the nucleus periphery towards its interior. We proved this concept by applying it to restriction enzyme diffusion, where we exploited a FISH-based method (YFISH) to visualize concentric genomic restriction signal waves generated by different digestion times. Specifically, GPSeq combines the sequencing of genomic loci restricted at different digestion time lengths into a so-called "GPSeq score," a reliable and accurate estimate of genomic loci centrality. We validated the GPSeq score against a collection of 68 DNA FISH probes, spanning 11 different chromosomes, data obtained from DamID-seq of Lamin B1, and also Hi-C chromatin contacts. Then, we utilized the radial maps drawn by GPSeq to reveal novel radial arrangements of different chromatin states and identify centrality predictors at different resolutions. Subsequently, we applied a novel 3D genome reconstruction algorithm to demonstrate how an additional centrality constraint can improve reconstructed structures’ quality. Specifically, 3D genome structures generated by a GPSeq-informed algorithm showed a higher correlation with FISH-based radial measurements and an arrangement of chromosome territories and genomic compartments that better reflects the underlying biology. Additionally, structures generated by the combination of GPSeq and Hi-C intrachromosomal contacts allowed the recovery of the inter-chromosomal contacts, further underscoring the necessity of additional constraints provided by orthogonal methods to Hi-C for a more reliable 3D genome reconstruction. Finally, we applied GPSeq to provide insight into the so-called "bodyguard hypothesis, " speculating that heterochromatin might act as a shield from exogenous mutagens for the more internally located active chromatin. In this regard, we showed that cancer-related single-nucleotide variants (SNVs) have a strikingly different radial arrangement than germline single-nucleotide polymorphisms (SNPs), with the former more peripherally located than the latter. We then showed that genomic regions involved with gene fusions in cancer tend to locate more internally and contact other chromosomes more frequently than other regions. We combined these observations and the fact that double-strand breaks (DSBs) tend to locate more internally, further confirmed from immunofluorescence experiments, to speculate that cancer-related SNVs and germline SNPs might come to be by different underlying mechanisms. Altogether, these results highlight the importance of genomewide high-resolution radial maps in the study of genome architecture, both as a standalone resource and as a complementary feature to chromatin contacts

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1’s intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.We thank the Montpellier Ressources Imagerie facility (BioCampus Montpellier, Centre National de la Recherche Scientifique [CNRS], INSERM, University of Montpellier) and for computer resources from CINECA (ISCRA grant thanks to computer resources from INFN and CINECA [ISCRA Grant HP10C8JWU7]). G.C., Q.S., and F.B. were supported by a grant from the European Research Council (Advanced Grant 3DEpi, 788972) and by the CNRS. This work was funded by EMBO and the Wellcome Trust (ALTF1554-2016 and 206475/Z/17/Z; to M.I.R.) as well as the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.)

Newborn Screening in Japan

“Newborn Screening in Japan—2021” is a topical collection of the International Journal of Neonatal Screening. Japan's newborn mass screening (NBS) was started in 1977 at the national level as a national project. Subsequently, screening was conducted for six diseases. From 2014 a tandem mass analyzer (tandem mass) was introduced nationwide, and in addition to the conventional amino acid metabolism disorders urea cycle disorders, organic acid metabolism disorders and fatty acid metabolism disorders have joined the target diseases. Screening is currently conducted for 20 diseases. The acceptance rate of mass screening in Japan is 100%, and top-level screening measures available in the world, such as a quality control system and an inspection system, are carried out. This book is an overview of the history, current status and future of NBS in Japan. I hope that readers are interested in this book

Investigating the 3D chromatin architecture with fluorescence microscopy

Chromatin is an assembly of DNA and nuclear proteins, which on the one hand has the function to properly store the 2 meters of DNA of a diploid human nucleus in a small volume and on the other hand regulates the accessibility of specific DNA segments for proteins. Many cellular processes like gene expression and DNA repair are affected by the three-dimensional architecture of chromatin. Cohesin is an important and well-studied protein that affects three-dimensional chromatin organization. One of the functions of this motor protein is the active generation of specific domain structures (topologically associating domains (TADs)) by the process of loop extrusion. Studies of cohesin depleted cells showed that TAD structures were lost on a population average. Due to this finding, the question arose, to what extent the functional nuclear architecture, that can be detected by confocal and structured illumination microscopy, is impaired when cells were cohesin depleted. The work presented in this thesis could show that the structuring of the nucleus in areas with different chromatin densities including the localization of important nuclear proteins as well as replication patterns was retained. Interestingly, cohesin depleted cells proceeded through an endomitosis leading to the formation of multilobulated nuclei. Obviously, important structural features of chromatin can form even in the absence of cohesin. In the here presented work, fluorescence microscopic methods were used throughout, and an innovative technique was developed, that allows flexible labeling of proteins with different fluorophores in fixed cells. With this technique DNA as well as peptide nucleic acid (PNA) oligonucleotides can be site-specifically coupled to antibodies via the Tub-tag technology and visualized by complementary fluorescently labeled oligonucleotides. The advantages and disadvantages of PNAs as docking strands are discussed in this thesis as well as the use of PNAs in fluorescence in situ hybridization (FISH). In the next study, which is part of this work, a combination of FISH and super-resolution microscopy was used. There it could be shown that DNA segments of 5 kb can form both compact and elongated configurations in regulatory active as well as inactive chromatin. Coarse-grained modeling of these microscopic data, in agreement with published data from other groups, has suggested that elongated configurations occur more frequently in DNA segments in which the occupancy of nucleosomes is reduced. The microscopically measured distance distributions could only be simulated with models that assume different densities of nucleosomes in the population. Another result of this study was that inactive chromatin - as expected - shows a high level of compaction, which can hardly be explained with common coarse-grained models. It is possible that environmental effects that are difficult to simulate play a role here. Chromatin is a highly dynamic structure, and its architecture is constantly changing, be it through active processes such as the effect of cohesin investigated here or through thermodynamic interactions of nucleosomes as they are simulated in coarse-grained models. It will take a long time until we adequately understand these dynamic processes and their interplay

Investigation into the Hydrothermal Treatment of Sugarcane Bagasse under Near- and Supercritical Conditions

Processing biomass in near- and supercritical water has garnered increasing attention because of its ability to accept a variety of wet feedstocks, energy efficiency, and ability to regulate the solubility and separation of components. Very few studies of biomass hydrothermal conversion provide a comprehensive evaluation of multiple process parameters and various additives and their effects on a single product phase. This research examined the influence of temperature, residence time, biomass concentration, and particle size on volatiles production from the hydrothermal conversion of sugarcane bagasse. Temperature had the greatest impact on volatile yields with the largest increase (23 wt %) occurring between 400 and 500 °C. The hydrogen mass yield increased 1000% between 300 and 600 °C. Increasing the residence time from 1 to 60 min resulted in a 49% increase in the mass yield of volatiles and 12.1% increase in the overall conversion of bagasse. The heating value of the volatile products declined after 10 min. Thermal cracking reactions dominated the early gas phase chemistry through 10 min but may have been accompanied by oxygenolysis of intermediate compounds at extended reaction intervals. In general, the use of Li/MgO and MnO2 catalysts improved the hydrothermal conversion of bagasse by 10%. An increased selectivity toward propylene production by both catalysts suggests metal oxide catalysts may promote partial oxidation via hydroxyl radicals. A side by side comparison of runs conducted in a Hastelloy X bomb and a titanium bomb revealed possible wall effects. The titanium bomb run produced 13 times more CO than the Hastelloy X bomb run along with at least 60 ppm of H2S. Lower hydrocarbon yields from the former run also support the theory that Ti is more catalytically active than Fe, Ni, and Cr in hydrothermal media. Chromatographic analysis revealed that methane in the volatile product generated from an experiment conducted using D2O instead of H2O had been perdeuterated, implying that hydrogen from the water medium is a labile participant in hydrothermal reactions. The Arrhenius parameters for bagasse hydrothermal conversion at 500 °C were determined as shown: Ea = 101.4 kJ•mol-1 and A = 1.28 × 109 min-1

CAF-1 p150 and Ki-67 Regulate Nuclear Structure Throughout the Human Cell Cycle

The three-dimensional organization of the human genome is non-random in interphase cells. Heterochromatin is highly clustered at the nuclear periphery, adjacent to nucleoli, and near centromeres. These localizations are reshuffled during mitosis when the chromosomes are condensed, nucleoli disassembled, and the nuclear envelope broken down. After cytokinesis, heterochromatin is re-localized to the domains described above. However, the mechanisms by which this localization is coordinated are not well understood. This dissertation will present evidence showing that both CAF-1 p150 and Ki-67 regulate nuclear structure throughout the human cell cycle. Chromatin Assembly Factor 1 (CAF-1) is a highly conserved three-subunit protein complex which deposits histones (H3/H4)2 heterotetramers onto replicating DNA during S-phase of the cell cycle. The N-terminal domain of the largest subunit of CAF-1 (p150N) is dispensable for histone deposition, and instead regulates the localization of specific loci (Nucleolar-Associated Domains, or “NADs”) and several proteins to the nucleolus during interphase. One of the proteins regulated by p150N is Ki-67, a protein widely used as a clinical marker of cellular proliferation. Depletion of Ki-67 decreases the association of NADs to the nucleolus in a manner similar to that of p150. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins that surrounds all condensed chromosomes during mitosis. A subset of p150 localizes to the PCL during mitosis, and depletion of p150 disrupts Ki-67 localization to the PCL. This activity was mapped to the Sumoylation Interacting Motif (SIM) within p150N, which is also required for the localization of NADs and Ki-67 to the nucleolus during interphase. Together, these studies indicate that p150N coordinates the three-dimensional arrangement of both interphase and mitotic chromosomes via Ki-67

Applications of next-generation technologies in the diagnosis of haematological diseases and cancer

The advent of massively-parallel next-generation sequencing (NGS) methods has provided researchers with a powerful tool with which to interrogate and characterise the molecular landscape of cancer genomes. Compared to existing methods of DNA sequencing, NGS platforms generate massive amounts of sequence data and, as a consequence, can reveal information not just on single nucleotide variations (SNVs), but also on copy-number aberrations, translocations and large insertions and deletions in a single experiment. Furthermore, targeted NGS provides the capability to focus on a small number of targets simultaneously, with high accuracy and sensitivity. The presence of specific molecular markers acts as predictors of disease outcome, survival rates and treatment response in individual patients. Screening for such markers has become routine practice in diagnostic laboratories using traditional methods of DNA analysis, are widely used in diagnostic laboratories around the world. Whilst these methods are proven and reliable, their limitations lie in the fact that they focus on only the most prevalent mutations in a particular cancer. The ability to investigate multiple gene targets within individual patients, to a high level of accuracy, and to monitor these changes over time will be a valuable tool in cancer diagnostics. As such, there is a potential use case for NGS techniques in routine diagnostics. Therefore, this thesis investigated the extent to which NGS platforms could be used in a clinical setting for the diagnosis and risk-stratification of both lymphoid and myeloid malignancies. A targeted next-generation sequencing panel was designed and validated against existing diagnostic methods. All mutations in the validation cohort were correctly identified. Both the specificity and sensitivity of the assay were determined and were considerably better than those of the current ‘gold-standard’ techniques. This panel has been fully validated and implemented into the diagnostic service at the John Radcliffe Hospital. The research applications of this panel were also demonstrated through the sequencing of a cohort of del(5q) MDS patients. It was not only found that mutations in TP53 and ASXL1 may be key drivers in the progression of del(5q) MDS into AML but also that 40% of del(5q) patients harboured at least one mutation. A number of mutations were below the limit of detection for Sanger sequencing, and so this study expands our knowledge of the del(5q) mutational landscape. Whole genome sequencing of 42 CLL cases revealed a high level of molecular heterogeneity, with mutations in key CLL driver genes including TP53, SF3B1, NOTCH1 and ATM. Both clinically relevant CNAs and translocations were detected in the cohort. Four mutation signatures were detected across the CLL genomes and are both associated with, and vary in their prevalence according to, specific clinical characteristics, including age and chemo-refractoriness. Mutations introduced as part of the SHM process in B-cells are present throughout the genome, including in patients with unmutated IgHV genes. Regions of localised hypermutation are present in CLL, with a number affecting genes associated with coding mutations in CLL, including ATM, KLHL6 and MEGF9. A number of mutation clusters are also identified in potentially regulatory regions of genes. In summary, this thesis demonstrates that both whole genome sequencing and targeted sequencing panels can be introduced into diagnostics to aid the clinical decision-making process and also reveal important new findings that increase our understanding of the pathogenesis of leukaemia