Undiagnosed RASopathies in infertile men.

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.

Prioritization of oligogenic variant combinations in whole exomes.

MOTIVATION: Whole exome sequencing (WES) has emerged as a powerful tool for genetic research, enabling the collection of a tremendous amount of data about human genetic variation. However, properly identifying which variants are causative of a genetic disease remains an important challenge, often due to the number of variants that need to be screened. Expanding the screening to combinations of variants in two or more genes, as would be required under the oligogenic inheritance model, simply blows this problem out of proportion. RESULTS: We present here the High-throughput oligogenic prioritizer (Hop), a novel prioritization method that uses direct oligogenic information at the variant, gene and gene pair level to detect digenic variant combinations in WES data. This method leverages information from a knowledge graph, together with specialized pathogenicity predictions in order to effectively rank variant combinations based on how likely they are to explain the patient's phenotype. The performance of Hop is evaluated in cross-validation on 36 120 synthetic exomes for training and 14 280 additional synthetic exomes for independent testing. Whereas the known pathogenic variant combinations are found in the top 20 in approximately 60% of the cross-validation exomes, 71% are found in the same ranking range when considering the independent set. These results provide a significant improvement over alternative approaches that depend simply on a monogenic assessment of pathogenicity, including early attempts for digenic ranking using monogenic pathogenicity scores. AVAILABILITY AND IMPLEMENTATION: Hop is available at https://github.com/oligogenic/HOP.

Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Faster and more accurate pathogenic combination predictions with VarCoPP2.0.

BACKGROUND: The prediction of potentially pathogenic variant combinations in patients remains a key task in the field of medical genetics for the understanding and detection of oligogenic/multilocus diseases. Models tailored towards such cases can help shorten the gap of missing diagnoses and can aid researchers in dealing with the high complexity of the derived data. The predictor VarCoPP (Variant Combinations Pathogenicity Predictor) that was published in 2019 and identified potentially pathogenic variant combinations in gene pairs (bilocus variant combinations), was the first important step in this direction. Despite its usefulness and applicability, several issues still remained that hindered a better performance, such as its False Positive (FP) rate, the quality of its training set and its complex architecture. RESULTS: We present VarCoPP2.0: the successor of VarCoPP that is a simplified, faster and more accurate predictive model identifying potentially pathogenic bilocus variant combinations. Results from cross-validation and on independent data sets reveal that VarCoPP2.0 has improved in terms of both sensitivity (95% in cross-validation and 98% during testing) and specificity (5% FP rate). At the same time, its running time shows a significant 150-fold decrease due to the selection of a simpler Balanced Random Forest model. Its positive training set now consists of variant combinations that are more confidently linked with evidence of pathogenicity, based on the confidence scores present in OLIDA, the Oligogenic Diseases Database ( https://olida.ibsquare.be ). The improvement of its performance is also attributed to a more careful selection of up-to-date features identified via an original wrapper method. We show that the combination of different variant and gene pair features together is important for predictions, highlighting the usefulness of integrating biological information at different levels. CONCLUSIONS: Through its improved performance and faster execution time, VarCoPP2.0 enables a more accurate analysis of larger data sets linked to oligogenic diseases. Users can access the ORVAL platform ( https://orval.ibsquare.be ) to apply VarCoPP2.0 on their data.

Toward reporting standards for the pathogenicity of variant combinations involved in multilocus/oligogenic diseases.

Although standards and guidelines for the interpretation of variants identified in genes that cause Mendelian disorders have been developed, this is not the case for more complex genetic models including variant combinations in multiple genes. During a large curation process conducted on 318 research articles presenting oligogenic variant combinations, we encountered several recurring issues concerning their proper reporting and pathogenicity assessment. These mainly concern the absence of strong evidence that refutes a monogenic model and the lack of a proper genetic and functional assessment of the joint effect of the involved variants. With the increasing accumulation of such cases, it has become essential to develop standards and guidelines on how these oligogenic/multilocus variant combinations should be interpreted, validated, and reported in order to provide high-quality data and supporting evidence to the scientific community.

Scaling up oligogenic diseases research with OLIDA: the Oligogenic Diseases Database.

Improving the understanding of the oligogenic nature of diseases requires access to high-quality, well-curated Findable, Accessible, Interoperable, Reusable (FAIR) data. Although first steps were taken with the development of the Digenic Diseases Database, leading to novel computational advancements to assist the field, these were also linked with a number of limitations, for instance, the ad hoc curation protocol and the inclusion of only digenic cases. The OLIgogenic diseases DAtabase (OLIDA) presents a novel, transparent and rigorous curation protocol, introducing a confidence scoring mechanism for the published oligogenic literature. The application of this protocol on the oligogenic literature generated a new repository containing 916 oligogenic variant combinations linked to 159 distinct diseases. Information extracted from the scientific literature is supplemented with current knowledge support obtained from public databases. Each entry is an oligogenic combination linked to a disease, labelled with a confidence score based on the level of genetic and functional evidence that supports its involvement in this disease. These scores allow users to assess the relevance and proof of pathogenicity of each oligogenic combination in the database, constituting markers for reporting improvements on disease-causing oligogenic variant combinations. OLIDA follows the FAIR principles, providing detailed documentation, easy data access through its application programming interface and website, use of unique identifiers and links to existing ontologies. DATABASE URL: https://olida.ibsquare.be.

Uterine sparing management in patients with endometrial cancer: a narrative literature review.

Endometrial cancer is the most common malignancy of the female genital tract. Approximately 25% of cases occur in premenopausal women, and up to 5% of cases occur in women who are younger than 40 years old. The survival rate in these cases is 99%; therefore, uterine-sparing management could be considered under strict criteria selection and the strong desire of the woman to preserve uterus and fertility. Diagnosis should be performed after a hysteroscopic biopsy instead of dilatation and curettage. The highest remission rate was achieved after combining a hysteroscopic resection with hormonal therapy compared to single hormonal treatment. The most common regiments are the following progestins: megestrol acetate (MA) and medroxyprogesterone acetate (MPA) taken orally with a daily dosage of 160 mg-320 mg for MA and 250 mg-600 mg for MP. Evaluations at three and six months could be performed by office endometrial biopsy and/or hysteroscopic directed biopsy especially in the presence of levonorgestrel intrauterine system, and in cases of remission, either a pregnancy attempt or maintenance therapy should be considered. After childbearing, hysterectomy with bilateral salpingo-oophorectomy is recommended, whereas ovarian preservation could be considered depending on the patient's age and whether they fulfil the strict criteria selection.

6th European Student Council Symposium (ESCS): overcoming obstacles to enhance virtuality, connectivity, inclusivity and community engagement.

This editorial summarises the organisation, activities, and scientific content of the 6th European Student Council Symposium (ESCS) organised by the International Society for Computational Biology Student Council (ISCB-SC). The event was held on September 6, 2020, as a satellite event preceding the ISCB's  19th European Conference in Computational Biology. Both events were first planned to be held in-person in Sitges, Spain, but moved virtually as a strategy to face the SARS-CoV2 sanitary crisis. This completely unforeseen situation has posed several challenges that have been successfully addressed thanks to the robust ISCB Student Council community structure and the strong commitment of the organisers. Despite all the obstacles and challenges, we have found that virtuality has several advantages that can continue to be kept to improve in-person meetings in the future and make conferences more inclusive allowing a larger audience to participate.

Digenic inheritance of human primary microcephaly delineates centrosomal and non-centrosomal pathways.

Primary microcephaly (PM) is characterized by a small head since birth and is vastly heterogeneous both genetically and phenotypically. While most cases are monogenic, genetic interactions between Aspm and Wdr62 have recently been described in a mouse model of PM. Here, we used two complementary, holistic in vivo approaches: high throughput DNA sequencing of multiple PM genes in human patients with PM, and genome-edited zebrafish modeling for the digenic inheritance of PM. Exomes of patients with PM showed a significant burden of variants in 75 PM genes, that persisted after removing monogenic causes of PM (e.g., biallelic pathogenic variants in CEP152). This observation was replicated in an independent cohort of patients with PM, where a PM gene panel showed in addition that the burden was carried by six centrosomal genes. Allelic frequencies were consistent with digenic inheritance. In zebrafish, non-centrosomal gene casc5 -/- produced a severe PM phenotype, that was not modified by centrosomal genes aspm or wdr62 invalidation. A digenic, quadriallelic PM phenotype was produced by aspm and wdr62. Our observations provide strong evidence for digenic inheritance of human PM, involving centrosomal genes. Absence of genetic interaction between casc5 and aspm or wdr62 further delineates centrosomal and non-centrosomal pathways in PM.

Automated Stopped-Flow Fluorimetric Sensor for Biologically Active Adamantane Derivatives Based on Zone Fluidics.

A zone-fluidics (ZF) based automated fluorimetric sensor for the determination of pharmaceutically active adamantine derivatives, i.e., amantadine (AMA), memantine (MEM) and rimantadine (RIM) is reported. Discrete zones of the analytes and reagents (o-phthalaldehyde and N-acetylcysteine) mix and react under stopped-flow conditions to yield fluorescent iso-indole derivatives (λex/ λem = 340/455 nm). The proposed ZF sensor was developed and validated to prove suitable for quality control tests (assay and content uniformity) of commercially available formulations purchased from the Greek market (EU licensed) and from non-EU web-pharmacies at a sampling rate of 16 h-1. Interestingly, a formulation obtained through the internet and produced in a third-non-EU-country (AMA capsules, 100 mg per cap), was found to be out of specifications (mean assay of 85.3%); a validated HPLC method was also applied for confirmatory purposes.

Using game theory and decision decomposition to effectively discern and characterise bi-locus diseases.

In order to gain insight into oligogenic disorders, understanding those involving bi-locus variant combinations appears to be key. In prior work, we showed that features at multiple biological scales can already be used to discriminate among two types, i.e. disorders involving true digenic and modifier combinations. The current study expands this machine learning work towards dual molecular diagnosis cases, providing a classifier able to effectively distinguish between these three types. To reach this goal and gain an in-depth understanding of the decision process, game theory and tree decomposition techniques are applied to random forest predictors to investigate the relevance of feature combinations in the prediction. A machine learning model with high discrimination capabilities was developed, effectively differentiating the three classes in a biologically meaningful manner. Combining prediction interpretation and statistical analysis, we propose a biologically meaningful characterization of each class relying on specific feature strengths. Figuring out how biological characteristics shift samples towards one of three classes provides clinically relevant insight into the underlying biological processes as well as the disease itself.

Global network of computational biology communities: ISCB's Regional Student Groups breaking barriers.

Regional Student Groups (RSGs) of the International Society for Computational Biology Student Council (ISCB-SC) have been instrumental to connect computational biologists globally and to create more awareness about bioinformatics education. This article highlights the initiatives carried out by the RSGs both nationally and internationally to strengthen the present and future of the bioinformatics community. Moreover, we discuss the future directions the organization will take and the challenges to advance further in the ISCB-SC main mission: "Nurture the new generation of computational biologists".

Diversity, Cyanotoxin Production, and Bioactivities of Cyanobacteria Isolated from Freshwaters of Greece.

Cyanobacteria are a diverse group of photosynthetic Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against a broad spectrum of organisms and cell lines. In this study, 29 strains isolated from freshwaters in Greece were classified using a polyphasic approach and assigned to Chroococcales, Synechococcales, and Nostocales, representing 11 genera and 17 taxa. There were good agreements between 16S ribosomal RNA (rRNA)-cpcBA-internal genetic spacer (IGS) characterization and morphological features, except for the Jaaginema-Limnothrix group which appears intermixed and needs further elucidation. Methanol extracts of the strains were analyzed for cyanotoxin production and tested against pathogenic bacteria species and several cancer cell lines. We report for the first time a Nostoc oryzae strain isolated from rice fields capable of producing microcystins (MCs) and a Chlorogloeopsis fritschii strain isolated from the plankton of a lake, suggesting that this species may also occur in freshwater temperate habitats. Strains with very high or identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activities. Extracts from Synechococcus cf. nidulans showed the most potent antibacterial activity against Staphylococcus aureus, whereas Jaaginema sp. strains exhibited potent cytotoxic activities against human colorectal adenocarcinoma and hepatocellular carcinoma cells. Jaaginema Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) 0110 and 0210 strains caused pronounced changes in the actin network and triggered the formation of numerous lipid droplets in hepatocellular carcinoma and green monkey kidney cells, suggesting oxidative stress and/or mitochondrial damage leading to apoptosis.

ORVAL: a novel platform for the prediction and exploration of disease-causing oligogenic variant combinations.

A tremendous amount of DNA sequencing data is being produced around the world with the ambition to capture in more detail the mechanisms underlying human diseases. While numerous bioinformatics tools exist that allow the discovery of causal variants in Mendelian diseases, little to no support is provided to do the same for variant combinations, an essential task for the discovery of the causes of oligogenic diseases. ORVAL (the Oligogenic Resource for Variant AnaLysis), which is presented here, provides an answer to this problem by focusing on generating networks of candidate pathogenic variant combinations in gene pairs, as opposed to isolated variants in unique genes. This online platform integrates innovative machine learning methods for combinatorial variant pathogenicity prediction with visualization techniques, offering several interactive and exploratory tools, such as pathogenic gene and protein interaction networks, a ranking of pathogenic gene pairs, as well as visual mappings of the cellular location and pathway information. ORVAL is the first web-based exploration platform dedicated to identifying networks of candidate pathogenic variant combinations with the sole ambition to help in uncovering oligogenic causes for patients that cannot rely on the classical disease analysis tools. ORVAL is available at https://orval.ibsquare.be.

Predicting disease-causing variant combinations.

Notwithstanding important advances in the context of single-variant pathogenicity identification, novel breakthroughs in discerning the origins of many rare diseases require methods able to identify more complex genetic models. We present here the Variant Combinations Pathogenicity Predictor (VarCoPP), a machine-learning approach that identifies pathogenic variant combinations in gene pairs (called digenic or bilocus variant combinations). We show that the results produced by this method are highly accurate and precise, an efficacy that is endorsed when validating the method on recently published independent disease-causing data. Confidence labels of 95% and 99% are identified, representing the probability of a bilocus combination being a true pathogenic result, providing geneticists with rational markers to evaluate the most relevant pathogenic combinations and limit the search space and time. Finally, the VarCoPP has been designed to act as an interpretable method that can provide explanations on why a bilocus combination is predicted as pathogenic and which biological information is important for that prediction. This work provides an important step toward the genetic understanding of rare diseases, paving the way to clinical knowledge and improved patient care.

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

KEY MESSAGE: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Smart Polymeric Nanoparticles as Emerging Tools for Imaging--The Parallel Evolution of Materials.

The field of imaging has developed considerably over the past decade and recent advances in the area of nanotechnology, in particular nanomaterials, have opened new opportunities. Polymeric nanoparticles are particularly interesting and a number of novel materials, characterized by stimuli-responsive characteristics and fluorescent tagging, have allowed visualization, intracellular labeling and real-time tracking. In some of the latest applications the nanoparticles have been used for imagining of tumor cells, both in vivo and ex vivo.

Development of PVP/PEG mixtures as appropriate carriers for the preparation of drug solid dispersions by melt mixing technique and optimization of dissolution using artificial neural networks.

The effect of plasticizer's (PEG) molecular weight (MW) on PVP based solid dispersions (SDs), prepared by melt mixing, was evaluated in the present study using Tibolone as a poorly water soluble model drug. PEGs with MW of 400, 600, and 2000 g/mol were tested, and the effect of drug content, time and temperature of melt mixing on the physical state of Tibolone, and the dissolution characteristics from SDs was investigated. PVP blends with PEG400 and PEG600 were completely miscible, while blends were heterogeneous. Furthermore, a single Tg recorded in all samples, indicating that Tibolone was dispersed in a molecular lever (or in the form of nanodispersions), varied with varying PEG's molecular weight, melt mixing temperature, and drug content, while FTIR analysis indicated significant interactions between Tibolone and PVP/PEG matrices. All prepared solid dispersion showed long-term physical stability (18 months in room temperature). The extent of interaction between mixture components was verified using Fox and Gordon-Taylor equations. Artificial neural networks, used to correlate the studied factors with selected dissolution characteristics, showed good prediction ability.

Novel core-shell magnetic nanoparticles for Taxol encapsulation in biodegradable and biocompatible block copolymers: preparation, characterization and release properties.

Theranostic polymeric nanocarriers loaded with anticancer drug Taxol and superparamagnetic iron oxide nanocrystals have been developed for possible magnetic resonance imaging (MRI) use and cancer therapy. Multifunctional nanocarriers with a core-shell structure have been prepared by coating superparamagnetic Fe3O4 nanoparticles with block copolymer of poly(ethylene glycol)-b-poly(propylene succinate) with variable molecular weights of the hydrophobic block poly(prolylene succinate). The multifunctional polymer nano-vehicles were prepared using a nanoprecipitation method. Scanning transmission electron microscopy revealed the encapsulation of magnetic nanoparticles inside the polymeric matrix. Energy dispersive X-ray spectroscopy and electron energy loss spectroscopy mapping allowed us to determine the presence of the different material ingredients in a quantitative way. The diameter of the nanoparticles is below 250 nm yielding satisfactory encapsulation efficiency. The nanoparticles exhibit a biphasic drug release pattern in vitro over 15 days depending on the molecular weight of the hydrophobic part of the polymer matrix. These new systems where anti-cancer therapeutics like Taxol and iron oxide nanoparticles (IOs) are co-encapsulated into new facile polymeric nanoparticles, could be addressed as potential multifunctional vehicles for simultaneous drug delivery and targeting imaging as well as real time monitoring of therapeutic effects.

Insight on the formation of chitosan nanoparticles through ionotropic gelation with tripolyphosphate.

This work reports details pertaining to the formation of chitosan nanoparticles that we prepare by the ionic gelation method. The molecular interactions of the ionic cross-linking of chitosan with tripolyphosphate have been investigated and elucidated by means of all-electron density functional theory. Solvent effects have been taken into account using implicit models. We have identified primary-interaction ionic cross-linking configurations that we define as H-link, T-link, and M-link, and we have quantified the corresponding interaction energies. H-links, which display high interaction energies and are also spatially broadly accessible, are the most probable cross-linking configurations. At close range, proton transfer has been identified, with maximum interaction energies ranging from 12.3 up to 68.3 kcal/mol depending on the protonation of the tripolyphosphate polyanion and the relative coordination of chitosan with tripolyphosphate. On the basis of our results for the linking types (interaction energies and torsion bias), we propose a simple mechanism for their impact on the chitosan/TPP nanoparticle formation process. We introduce the ß ratio, which is derived from the commonly used α ratio but is more fundamental since it additionally takes into account structural details of the oligomers.