Juvenile Primary Sjögren Syndrome in a 15-Year-Old Boy with Renal Involvement: A Case Report and Review of the Literature.

Juvenile primary Sjögren syndrome (pSS) with renal involvement is extremely rare, reported approximately in 50 children, predominantly girls. Here, we present the first reported case of a male child with juvenile pSS with ocular surface disease (previously keratoconjunctivitis sicca), submandibular salivary gland involvement, and tubulointerstitial nephritis. First, two symptoms were clinically apparent at presentation. We illustrate here that kidney involvement in pSS should be actively looked for, as juvenile pSS may be associated with asymptomatic renal involvement. Immunophenotyping of peripheral blood cells using multicolor flow cytometry revealed at the time of diagnosis changes in both adaptive (T memory cells and B memory cells), and innate immunity (an increased activation of natural killer cells, as well as monocytes and neutrophils, and an increased representation of intermediate monocytes). Our case report points to the importance of kidney examination, early diagnosis and therapy in juvenile pSS, as well as highlights international collaboration to obtain more data for this rare disease.

Determining Thrombogenicity: Using a Modified Thrombin Generation Assay to Detect the Level of Thrombotic Event Risk in Lupus Anticoagulant-Positive Patients.

The aim of this study was to determine the thrombogenicity of lupus anticoagulant (LA) antibodies using a modified thrombin generation assay (TGA) with the addition of activated protein C (APC) in a group of 85 patients with LA-positive samples. Of these, 58 patients had clinical manifestations of antiphospholipid syndrome (APS) according to the Sydney criteria classification, i.e., each patient had thrombosis or foetal loss, and 27 patients did not show any clinical manifestations of APS. A comparison of the two groups' TGA results revealed statistically significant differences (Fisher's test p = 0.0016). The group of patients exhibiting clinical manifestations of APS showed higher thrombogenicity in 56.9% of patients, while the group of patients not yet exhibiting clinical manifestations of APS showed higher thrombogenicity in 25.9% of patients. There were no significant differences in the specificity of the TGA test between the groups of patients exhibiting similar clinical manifestations. Receiver operating characteristic curve analysis showed a more significant relationship (p = 0.0060) for TGA than for LA titre (p = 0.3387). These data suggest that the determination of LA thrombogenicity with the TGA assay leads to an increased prediction of the manifestation of a thromboembolic event. Our findings appear to be particularly relevant for the prediction of thrombotic events in patients with laboratory-expressed APS and no clinical manifestations.

Long-term environmental metal exposure is associated with hypomethylation of CpG sites in NFKB1 and other genes related to oncogenesis.

BACKGROUND: Long-term environmental exposure to metals leads to epigenetic changes and may increase risks to human health. The relationship between the type and level of metal exposure and epigenetic changes in subjects exposed to high concentrations of metals in the environment is not yet clear. The aim of our study is to find the possible association of environmental long-term exposure to metals with DNA methylation changes of genes related to immune response and carcinogenesis. We investigated the association of plasma levels of 21 essential and non-essential metals detected by ICP-MS and the methylation level of 654 CpG sites located on NFKB1, CDKN2A, ESR1, APOA5, IGF2 and H19 genes assessed by targeted bisulfite sequencing in a cohort of 40 subjects living near metal mining area and 40 unexposed subjects. Linear regression was conducted to find differentially methylated positions with adjustment for gender, age, BMI class, smoking and metal concentration. RESULTS: In the metal-exposed group, five CpGs in the NFKB1 promoter region were hypomethylated compared to unexposed group. Four differentially methylated positions (DMPs) were associated with multiple metals, two of them are located on NFKB1 gene, and one each on CDKN2A gene and ESR1 gene. Two DMPs located on NFKB1 (chr4:102500951, associated with Be) and IGF2 (chr11:2134198, associated with U) are associated with specific metal levels. The methylation status of the seven CpGs located on NFKB1 (3), ESR1 (2) and CDKN2A (2) positively correlated with plasma levels of seven metals (As, Sb, Zn, Ni, U, I and Mn). CONCLUSIONS: Our study revealed methylation changes in NFKB1, CDKN2A, IGF2 and ESR1 genes in individuals with long-term human exposure to metals. Further studies are needed to clarify the effect of environmental metal exposure on epigenetic mechanisms and pathways involved.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

Deep Molecular Response Achieved with Chemotherapy, Dasatinib and Interferon α in Patients with Lymphoid Blast Crisis of Chronic Myeloid Leukaemia.

The treatment outcome in patients with chronic myeloid leukaemia (CML) in blast crisis (BC) is unsatisfactory despite the use of allogeneic stem cell transplantation (ASCT). Moreover, in some patients ASCT is contraindicated, with limited treatment options. We report the case series of two patients with lymphoid BC CML in whom ASCT was not approachable. The first patient developed BC two months after diagnosis in association with dic(7;9)(p11.2;p11.2) and T315I mutation. Blast crisis with central nervous system leukemic involvement and K611N mutation of the SETD2 gene developed abruptly in the second patient five years after ceasing treatment with nilotinib in major molecular response (MMR) at the patient's request. Both underwent one course of chemotherapy in combination with rituximab and imatinib, followed by dasatinib and interferon α (INFα) treatment in the first and dasatinib alone in the second case. Deep molecular response (DMR; MR 4.0) was achieved within a short time in both cases. It is probable that DMR was caused by a specific immune response to CML cells, described in both agents. The challenging medical condition that prompted these case series, and the subsequent results, suggest a re-visit to the use of a combination of well-known drugs as an area for further investigation.

Antiphospholipid antibody-mediated NK cell cytotoxicity.

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia that is characterised by thrombosis and obstetric complications in the presence of antiphospholipid antibodies (aPL). Pregnancy complications remain a challenging problem for patients with APS, especially during the first trimester. Although natural killer (NK) cells constitute up to 70% of decidual lymphocytes during the first trimester, their contribution to early pregnancy loss in APS is largely unknown. We aimed to analyse whether aPL are able to recruit antibody-dependent cellular cytotoxicity (ADCC) of NK cells, with special emphasis on the differences in the effects of aPL containing anti-ß2GPI domain 1 (anti-ß2GPI-D1) antibodies (aPL+/D1+) and those that do not (aPL+/D1-). Our findings revealed a differential distribution of NK subsets in the presence of different aPL. Namely, aPL+/D1- IgGs increased CD56dim/CD16dim cells, while aPL+/D1 + IgGs increased the number of CD56bright/CD16dim cells. ADCC NK cell cytotoxicity was found to be higher in the presence of aPL+/D1- IgGs, as defined by an increased target cell death, degranulation and increased expression of CD11b, CD69 and NKG2D. Overall, our evidence showed that aPL are able to recruit ADCC, suggesting NK cells as candidate cells for APS-related obstetric complications.

Cellular senescence marker p16INK4a and NFKB1 gene polymorphisms in lower gastro-intestinal acute graft versus host disease.

BACKGROUND: Lower gastrointestinal (GI) graft versus host disease (GVHD) represents a severe complication in allogeneic hematopoietic stem cell transplant (HSCT) recipients with high rates of transplant-related mortality. Deregulated innate immunity reactions are the features of its pathogenesis. Cellular senescence has been considered a program of the innate immunity. We focused on lower GI GVHD from the perspective of cellular senescence. OBJECTIVE: We analyzed the impact of p16INK4a expression, a hallmark of cellular senescence, in intestinal biopsies of patients with lower GI GVHD symptoms and NFKB1 gene polymorphisms (rs3774937 C/T and rs3774959 A/G) on HSCT outcome. STUDY DESIGN: Fifty-two single-center patients who presented with symptoms of lower GI GVHD were analyzed in a retrospective manner. Two SNPs located in the NFKB1 gene regions (rs3774937 C/T and rs3774959 A/G) were genotyped from the peripheral blood samples collected before the start of the conditioning. All patients underwent proctosigmoidoscopy with biopsy of the mucosa. The expression of p16INK4a was analyzed in normal intestinal crypts and stroma. RESULTS: Fifty-two patients (50% male) received HSCT for hematological diseases (acute leukemias in 67%) and developed lower GI symptoms. Patients with p16INK4a expression in the intestinal stroma were in lower risk of developing histological grade 3-4 aGVHD (RR 0.18 [95% CI 0.05-0.65]; p = 0.009). The multivariate linear regression confirmed the independent effect of p16INK4a expression on time of the lower GI aGVHD symptoms onset (Coef. 38.9 [95% CI 12.7-65.1]; p = 0.005). The NFKB1 rs3774937 CC and TT/TC genotype were present in 40 and 80% of patients with p16INK4a expression, respectively (p = 0.04). The rs3774959 AA and GG/AG genotype were present among 43 and 82% of patients with p16INK4a expression, respectively (p = 0.02). Expression of p16INK4a was associated with no clinical variable but NFKB1 genotype. CONCLUSIONS: Our results address possible new mechanisms that may lead to better understanding of HSCT-related immune complications. Cellular senescence may bring novel approaches in GVHD diagnostics and therapy.

Network Analysis for Uncovering the Relationship between Host Response and Clinical Factors to Virus Pathogen: Lessons from SARS-CoV-2.

Analysing complex datasets while maintaining the interpretability and explainability of outcomes for clinicians and patients is challenging, not only in viral infections. These datasets often include a variety of heterogeneous clinical, demographic, laboratory, and personal data, and it is not a single factor but a combination of multiple factors that contribute to patient characterisation and host response. Therefore, multivariate approaches are needed to analyse these complex patient datasets, which are impossible to analyse with univariate comparisons (e.g., one immune cell subset versus one clinical factor). Using a SARS-CoV-2 infection as an example, we employed a patient similarity network (PSN) approach to assess the relationship between host immune factors and the clinical course of infection and performed visualisation and data interpretation. A PSN analysis of ~85 immunological (cellular and humoral) and ~70 clinical factors in 250 recruited patients with coronavirus disease (COVID-19) who were sampled four to eight weeks after a PCR-confirmed SARS-CoV-2 infection identified a minimal immune signature, as well as clinical and laboratory factors strongly associated with disease severity. Our study demonstrates the benefits of implementing multivariate network approaches to identify relevant factors and visualise their relationships in a SARS-CoV-2 infection, but the model is generally applicable to any complex dataset.

Determination of Thrombogenicity Levels of Various Antiphospholipid Antibodies by a Modified Thrombin Generation Assay in Patients with Suspected Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a hypercoagulable state accompanied by the presence of heterogeneous antiphospholipid antibodies (aPL), which nonspecifically affect hemostasis by the presence of lupus anticoagulans (LA), anticardiolipin antibodies (aCL), antibodies against ß2-glycoprotein-I (anti-ß2GPI), but also non-criteria antibodies such as antibodies against ß2-glycoprotein-I domain I (anti-DI), anti-phosphatidylserine/prothrombin (anti-PS/PT), anti-annexin V, and many others. The main target of the antibodies is the activated protein C (APC) system, the elimination of which can manifest itself as a thrombotic complication. The aim of this study was to determine the thrombogenicity of antibodies using a modified protein C-activated thrombin generation assay (TGA) on a group of 175 samples suspected of APS. TGA was measured with/without APC and the ratio of both measurements was evaluated (as for APC resistance), where a cut-off was calculated ≤4.5 (90th percentile) using 21 patients with heterozygous factor V Leiden mutation (FV Leiden heterozygous). Our study demonstrates the well-known fact that multiple positivity of different aPLs is a more severe risk for thrombosis than single positivity. Of the single antibody positivity, LA antibodies are the most serious (p value < 0.01), followed by aCL and their subgroup anti-DI (p value < 0.05). Non-criteria antibodies anti-annexin V and anti-PT/PS has a similar frequency occurrence of thrombogenicity as LA antibodies but without statistical significance or anti-ß2GPI1 positivity. The modified TGA test can help us identify patients in all groups who are also at risk for recurrent thrombotic and pregnancy complications; thus, long-term prophylactic treatment is appropriate. For this reason, it is proving increasingly beneficial to include the determination antibodies in combination with modified TGA test.

Rare germline ATM variants of uncertain significance in chronic lymphocytic leukaemia and other cancers.

Germline pathogenic ATM (ataxia-telangiectasia mutated) variants are associated with the risk of multiple cancers; however, genetic testing reveals a large number of ATM variants of uncertain significance (VUS). Here, we studied germline ATM variants occurring in a real-world cohort of 336 patients with chronic lymphocytic leukaemia (CLL) and public cancer whole-exome/genome-sequencing datasets (445 CLL, 75 mantle cell lymphoma, 216 metastatic breast cancer, 140 lung cancer patients). We found that two-thirds of rare germline ATM variants are pathogenic (18%-50%) or VUS-predicted pathogenic (50%-82%), depending on cancer type and reaching a prevalence of up to 8%, and one-third are VUS-predicted benign. Patients with both pathogenic and VUS-predicted pathogenic variants, all heterozygous, mostly missense, are more predisposed to biallelic ATM inactivation by acquiring deletion (del)11q than patients without these variants, similar to patients with somatic ATM variants. A functional assay of ATM activity in primary CLL cells proved that VUS-predicted pathogenic ATM variants partially reduce ATM activity and concurrent del(11q) leads to complete loss of ATM activity. The rare germline variants were associated with reduced progression-free survival in CLL on novel agents, comparable to somatic ATM or TP53 disruptions. Our results highlight the need to determine the pathogenicity of VUS in clinically relevant genes such as ATM.

N-acetylcysteine Can Induce Massive Oxidative Stress, Resulting in Cell Death with Apoptotic Features in Human Leukemia Cells.

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities.

Chronic lymphocytic leukaemia (CLL) is a genetically, morphologically and phenotypically heterogeneous chronic disease with clinical variability between patients. Whether the significant heterogeneity of cell size within the CLL population contributes to the heterogeneous features of this disease has not been investigated. The present study aimed to characterise the phenotypic and functional properties of two subpopulations of typical CLL cells that differ in cell size: small (s-CLL) and large (l-CLL) CLL cells delineated by forward scatter cytometry. The s-CLL cells were characterised by the CD5lowCXCR4hi phenotype, while the l-CLL cells were characterised by the CD5hiCXCR4dim phenotype and indicated a higher expression of CXCR3, CD20, CD38 and HLA-DR. The l-CLL cells displayed higher migration activity towards CXCL12, a tendency towards a higher proliferation rate and an increased capacity to produce IgM in the presence of CpG compared with s-CLL cells. When stimulated with CpG and CXCL12, l-CLL cells were characterised by a higher polarisation phenotype and motility than s-CLL cells. Our study revealed that the differences in CLL cell size reflected their activation status, polarisation and migratory abilities. Our data provide evidence of the importance of cell-size heterogeneity within a CLL pool and the dynamics of cell-size changes for disease pathogenesis, thus deserving further investigation.

DSP rs2076295 variants influence nintedanib and pirfenidone outcomes in idiopathic pulmonary fibrosis: a pilot study.

BACKGROUND: The antifibrotic drugs nintedanib and pirfenidone are used for the treatment of idiopathic pulmonary fibrosis (IPF). We analysed the association of common profibrotic polymorphisms in MUC5B (mucin 5B, rs35705950) and DSP (desmoplakin, rs2076295) on antifibrotic treatment outcomes in IPF. METHODS: MUC5B rs35705950 and DSP rs2076295 were assessed in IPF patients (n = 210, 139 men/71 women) from the Czech EMPIRE registry and age- or sex-matched healthy individuals (n = 205, 125 men/80 women). Genetic data were collated with overall survival (OS), acute exacerbation episodes, worsening lung function and antifibrotic treatment. RESULTS: We confirmed overexpression of the MUC5B rs35705950*T allele (55.2% versus 20.9%, p < 0.001) and the DSP rs2076295*G allele (80.4% versus 68.3%, p < 0.001) in IPF compared with controls. On antifibrotic drugs, lower mortability was observed in IPF patients with DSP G* allele (p = 0.016) and MUC5B T* allele (p = 0.079). Carriers of the DSP rs2076295*G allele benefitted from nintedanib treatment compared with TT genotype by a longer OS [hazard ratio (HR) = 7.99; 95% confidence interval (CI) = 1.56-40.90; p = 0.013] and a slower decline in lung function (HR = 8.51; 95% CI = 1.68-43.14; p = 0.010). Patients with a TT genotype (rs2076295) benefitted from treatment with pirfenidone by prolonged OS (p = 0.040; HR = 0.35; 95% CI = 0.13-0.95) compared with nintedanib treatment. Both associations were confirmed by cross-validation analysis. After stratifying by MUC5B rs35705950*T allele carriage, no difference in treatment outcome was observed for nintedanib or pirfenidone (p = 0.784). In the multivariate model, smoking, age, forced vital capacity (FVC) and DLCO (diffuse lung capacity) at the IPF diagnosis were associated with survival. CONCLUSION: Our real-world study showed that IPF patients with MUC5B T* allele or DSP G* allele profit from antifibrotic treatment by lower mortability. Moreover, carriers of the DSP rs2076295*G allele benefit from treatment with nintedanib, and TT genotype from treatment with pirfenidone. MUC5B rs35705950 did not impact the outcome of treatment with either nintedanib or pirfenidone. Our single-registry pilot study should be confirmed with an independent patient cohort.

Distribution of SARS-CoV-2 Lineages in the Czech Republic, Analysis of Data from the First Year of the Pandemic.

In the Czech Republic, the current pandemic led to over 1.67 million SARS-CoV-2- positive cases since the recording of the first case on 1 March 2020. SARS-CoV-2 genome analysis is an important tool for effective real-time quantitative PCR (RT-qPCR) diagnostics, epidemiology monitoring, as well as vaccination strategy. To date, there is no comprehensive report on the distribution of SARS-CoV-2 genome variants in either the Czech Republic, including Central and Eastern Europe in general, during the first year of pandemic. In this study, we have analysed a representative cohort of SARS-CoV-2 genomes from 229 nasopharyngeal swabs of COVID-19 positive patients collected between March 2020 and February 2021 using validated reference-based sequencing workflow. We document the changing frequency of dominant variants of SARS-CoV-2 (from B.1 -> B.1.1.266 -> B.1.258 -> B.1.1.7) throughout the first year of the pandemic and list specific variants that could impact the diagnostic efficiency RT-qPCR assays. Moreover, our reference-based workflow provided evidence of superinfection in several samples, which may have contributed to one of the highest per capita numbers of COVID-19 cases and deaths during the first year of the pandemic in the Czech Republic.

Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities.

Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp-50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.

Excellent option for mass testing during the SARS-CoV-2 pandemic: painless self-collection and direct RT-qPCR.

The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Of these subjects, 28.6% tested positive for SARS-CoV-2 using nasopharyngeal swab sampling. Our direct RT-qPCR approach for self-collected nasal swabs performed well with results similar to those of the two-step RT-qPCR on RNA isolates, achieving 0.99 positive and 0.98 negative predictive values (cycle threshold [Ct] < 37). Our research also reports on grey-zone viraemia, including samples with near-cut-off Ct values (Ct ≥ 37). In all investigated subjects (n = 20) with grey-zone viraemia, the ultra-small viral load disappeared within hours or days with no symptoms. Overall, this study underscores the importance of painless nasal-swab self-collection and direct RT-qPCR for mass testing during the SARS-CoV-2 pandemic and in the post-pandemic era.

Distribution of Merlin in eukaryotes and first report of DNA transposons in kinetoplastid protists.

DNA transposons are defined as repeated DNA sequences that can move within the host genome through the action of transposases. The transposon superfamily Merlin was originally found mainly in animal genomes. Here, we describe a global distribution of the Merlin in animals, fungi, plants and protists, reporting for the first time their presence in Rhodophyceae, Metamonada, Discoba and Alveolata. We identified a great variety of potentially active Merlin families, some containing highly imperfect terminal inverted repeats and internal tandem repeats. Merlin-related sequences with no evidence of mobilization capacity were also observed and may be products of domestication. The evolutionary trees support that Merlin is likely an ancient superfamily, with early events of diversification and secondary losses, although repeated re-invasions probably occurred in some groups, which would explain its diversity and discontinuous distribution. We cannot rule out the possibility that the Merlin superfamily is the product of multiple horizontal transfers of related prokaryotic insertion sequences. Moreover, this is the first account of a DNA transposon in kinetoplastid flagellates, with conserved Merlin transposase identified in Bodo saltans and Perkinsela sp., whereas it is absent in trypanosomatids. Based on the level of conservation of the transposase and overlaps of putative open reading frames with Merlin, we propose that in protists it may serve as a raw material for gene emergence.

Comparison of structural variants detected by optical mapping with long-read next-generation sequencing.

MOTIVATION: Recent studies have shown the potential of using long-read whole-genome sequencing (WGS) approaches and optical mapping (OM) for the detection of clinically relevant structural variants (SVs) in cancer research. Three main long-read WGS platforms are currently in use: Pacific Biosciences (PacBio), Oxford Nanopore Technologies (ONT) and 10x Genomics. Recently, whole-genome OM technology (Bionano Genomics) has been introduced into human diagnostics. Questions remain about the accuracy of these long-read sequencing platforms, how comparable/interchangeable they are when searching for SVs and to what extent they can be replaced or supplemented by OM. Moreover, no tool can effectively compare SVs obtained by OM and WGS. RESULTS: This study compared optical maps of the breast cancer cell line SKBR3 with AnnotSV outputs from WGS platforms. For this purpose, a software tool with comparative and filtering features was developed. The majority of SVs up to a 50 kbp distance variance threshold found by OM were confirmed by all WGS platforms, and ∼99% of translocations and ∼80% of deletions found by OM were confirmed by both PacBio and ONT, with ∼70% being confirmed by 10x Genomics in combination with PacBio and/or ONT. Interestingly, long deletions (>100 kbp) were detected only by 10x Genomics. Regarding insertions, ∼74% was confirmed by PacBio and ONT, but none by 10x Genomics. Inversions and duplications detected by OM were not detected by WGS. Moreover, the tool enabled the confirmation of SVs that overlapped in the same gene(s) and was applied to the filtering of disease-associated SVs. AVAILABILITY AND IMPLEMENTATION: https://github.com/novosadt/om-annotsv-svc.

Determining optical mapping errors by simulations.

MOTIVATION: Optical mapping is a complementary technology to traditional DNA sequencing technologies, such as next-generation sequencing (NGS). It provides genome-wide, high-resolution restriction maps from single, stained molecules of DNA. It can be used to detect large and small structural variants, copy number variations and complex rearrangements. Optical mapping is affected by different kinds of errors in comparison with traditional DNA sequencing technologies. It is important to understand the source of these errors and how they affect the obtained data. This article proposes a novel approach to modeling errors in the data obtained from the Bionano Genomics Inc. Saphyr system with Direct Label and Stain (DLS) chemistry. Some studies have already addressed this issue for older instruments with nicking enzymes, but we are unaware of a study that addresses this new system. RESULTS: The main result is a framework for studying errors in the data obtained from the Saphyr instrument with DLS chemistry. The framework's main component is a simulation that computes how major sources of errors for this instrument (a false site, a missing site and resolution errors) affect the distribution of fragment lengths in optical maps. The simulation is parametrized by variables describing these errors and we are using a differential evolution algorithm to evaluate parameters that best fit the data from the instrument. Results of the experiments manifest that this approach can be used to study errors in the optical mapping data analysis. AVAILABILITY AND IMPLEMENTATION: Source codes supporting the presented results are available at: https://github.com/mvasinek/olgen-om-error-prediction. The data underlying this article are available on the Bionano Genomics Inc. website, at: https://bionanogenomics.com/library/datasets/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.