eDAVE - Extension of GDC data analysis, visualization, and exploration tools.

Publicly available repositories such as Genomic Data Commons or Gene Expression Omnibus are a valuable research resource useful for hypothesis driven research as well as validation of the results of new experiments. Frequently however, the use of those opulent resources is challenging because advanced computational skills are required to mine deposited data. To address this challenge, we have developed eDAVE, a user-friendly, web and desktop interface enabling intuitive and robust analysis of almost 12 000 methylomes and transcriptomes from over 200 types of cells and tissues deposited in the Genomic Data Commons repository. The application is implemented in Python, supported for major browsers and available at: https://edave.pum.edu.pl/.

Identified in blood diet-related methylation changes stratify liver biopsies of NAFLD patients according to fibrosis grade.

BACKGROUND: High caloric diet and lack of physical activity are considered main causes of NAFLD, and a change in the diet is still the only effective treatment of this disease. However, molecular mechanism of the effectiveness of diet change in treatment of NAFLD is poorly understood. We aimed to assess the involvement of epigenetic mechanisms of gene expression regulation in treatment of NAFLD. Eighteen participants with medium- to high-grade steatosis were recruited and trained to follow the Mediterranean diet modified to include fibre supplements. At three timepoints (baseline, after 30 and 60 days), we evaluated adherence to the diet and measured a number of physiological parameters such as anthropometry, blood and stool biochemistry, liver steatosis and stiffness. We also collected whole blood samples for genome-wide methylation profiling and histone acetylation assessment. RESULTS: The diet change resulted in a decrease in liver steatosis along with statistically significant, but a minor change in BMI and weight of our study participants. The epigenetic profiling of blood cells identified significant genome-wide changes of methylation and acetylation with the former not involving regions directly regulating gene expression. Most importantly, we were able to show that identified blood methylation changes occur also in liver cells of NAFLD patients and the machine learning-based classifier that we build on those methylation changes was able to predict the stage of liver fibrosis with ROC AUC = 0.9834. CONCLUSION: Methylomes of blood cells from NAFLD patients display a number of changes that are most likely a consequence of unhealthy diet, and the diet change appears to reverse those epigenetic changes. Moreover, the methylation status at CpG sites undergoing diet-related methylation change in blood cells stratifies liver biopsies from NAFLD patients according to fibrosis grade.

Epigenetic activation of antiviral sensors and effectors of interferon response pathways during SARS-CoV-2 infection.

Recent studies have shown that methylation changes identified in blood cells of COVID-19 patients have a potential to be used as biomarkers of SARS-CoV-2 infection outcomes. However, different studies have reported different subsets of epigenetic lesions that stratify patients according to the severity of infection symptoms, and more importantly, the significance of those epigenetic changes in the pathology of the infection is still not clear. We used methylomics and transcriptomics data from the largest so far cohort of COVID-19 patients from four geographically distant populations, to identify casual interactions of blood cells' methylome in pathology of the COVID-19 disease. We identified a subset of methylation changes that is uniformly present in all COVID-19 patients regardless of symptoms. Those changes are not present in patients suffering from upper respiratory tract infections with symptoms similar to COVID-19. Most importantly, the identified epigenetic changes affect the expression of genes involved in interferon response pathways and the expression of those genes differs between patients admitted to intensive care units and only hospitalized. In conclusion, the DNA methylation changes involved in pathophysiology of SARS-CoV-2 infection, which are specific to COVID-19 patients, can not only be utilized as biomarkers in the disease management but also present a potential treatment target.

Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing.

Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

Long-Term Treatment with Bortezomib Induces Specific Methylation Changes in Differentiated Neuronal Cells.

Bortezomib (BTZ) is proteasome inhibitor, effectively used in the treatment of multiple myeloma, but frequently discontinued due to peripheral neuropathy, which develops in patients after consecutive treatment cycles. The molecular mechanisms affected by BTZ in neuronal cells, which result in neuropathy, remain unknown. However, BTZ is unlikely to lead to permanent morphological nerve damage, because neuropathy reverses after discontinuation of treatment, and nerve cells have very limited renewal capacity. We have previously shown that BTZ induces methylation changes in SH-SY5Y cells, which take part in the development of treatment resistance. Here, we hypothesized that BTZ affects the methylomes of mature neurons, and these changes are associated with BTZ neurotoxicity. Thus, we studied methylomes of neuronal cells, differentiated from the LUHMES cell line, after cycles of treatment with BTZ. Our results show that BTZ induces specific methylation changes in mature neurons, which are not present in SH-SY5Y cells after BTZ treatment. These changes appear to affect genes involved in morphogenesis, neurogenesis, and neurotransmission. Furthermore, identified methylation changes are significantly enriched within binding sites of transcription factors previously linked to neuron physiology, including EBF, PAX, DLX, LHX, and HNF family members. Altogether, our results indicate that methylation changes are likely to be involved in BTZ neurotoxicity.

Bortezomib induces methylation changes in neuroblastoma cells that appear to play a significant role in resistance development to this compound.

The anticancer activity of bortezomib (BTZ) has been increasingly studied in a number of indications and promising results for the use of this treatment have been shown in neuroblastoma. As BTZ treatment is usually administered in cycles, the development of resistance and side effects in patients undergoing therapy with BTZ remains a major challenge for the clinical usage of this compound. Common resistance development also means that certain cells are able to survive BTZ treatment and bypass molecular mechanisms that render BTZ anticancer activity. We studied the methylome of neuroblastoma cells that survived BTZ treatment. Our results indicate that BTZ induces pronounced genome wide methylation changes in cells which recovered from the treatment. Functional analyses of identified methylation changes demonstrated they were involved in key cancer pathology pathways. These changes may allow the cells to bypass the primary anticancer activity of BTZ and develop a treatment resistant and proliferative phenotype. To study whether cells surviving BTZ treatment acquire a proliferative phenotype, we repeatedly treated cells which recovered from the first round of BTZ treatment. The repetitive treatment led to induction of the extraordinary proliferative potential of the cells, that increased with subsequent treatments. As we did not observe similar effects in cells that survived treatment with lenalidomide, and non-treated cells cultured under the same experimental conditions, this phenomenon seems to be BTZ specific. Overall, our results indicate that methylation changes may play major role in the development of BTZ resistance.

Short history of 5-methylcytosine: from discovery to clinical applications.

Covalent modifications of nucleotides in genetic material have been known from the beginning of the last century. Currently, one of those modifications referred to as DNA methylation, is impacting personalised medicine both as a treatment target and a biomarker source for clinical disease management. In this short review, we describe landmark discoveries that led to the elucidation of the DNA methylation importance in the cell's physiology and clarification of its role as one of the major processes in disease pathology. We also describe turning points in the development of methodologies to study this modification, which ultimately resulted in the development of in-vitro diagnostic kits targeting disease related DNA methylation changes as biomarkers.

Correction to: Current status of development of methylation biomarkers for in vitro diagnostic IVD applications.

An amendment to this paper has been published and can be accessed via the original article.

Current status of development of methylation biomarkers for in vitro diagnostic IVD applications.

A significant volume of research clearly shows that disease-related methylation changes can be used as biomarkers at all stages of clinical disease management, including risk assessment and predisposition screening through early diagnostics to personalization of patient care and monitoring of the relapse and chronic disease. Thus disease-related methylation changes are an attractive source of the biomarkers that can have significant impact on precision medicine. However, the translation of the research findings in methylation biomarkers field to clinical practice is at the very least not satisfactory. That is mainly because the evidence generated in research studies indicating the utility of the disease-related methylation change to predict clinical outcome is in majority of the cases not sufficient to postulate the diagnostic use of the biomarker. The research studies need to be followed by well-designed and systematic investigations of clinical utility of the biomarker that produce data of sufficient quality to meet regulatory approval for the test to be used to make clinically valid decision. In this review, we describe methylation-based IVD tests currently approved for IVD use or at the advanced stages of the development for the diagnostic use. For each of those tests, we analyze the technologies that the test utilizes for methylation detection as well as describe the types of the clinical studies that were performed to show clinical validity of the test and warrant regulatory approval. The examples reviewed here should help with planning of clinical investigations and delivery of the clinical evidence required for the regulatory approval of potential methylation biomarker based IVD tests.

Relationship between toll-like receptor 2 R753Q and T16934A polymorphisms and Staphylococcus aureus nasal carriage.

BACKGROUND: The association among specific single-nucleotide polymorphisms (SNPs) in TLR2 R753Q (rs5743708) and T16934A (rs4696480) and the nasal carriage of Staphylococcus aureus was studied in adults before CABG. METHODS: The TLR2 polymorphisms were genotyped in 299 consecutive patients prepared for a CABG operation. Genotyping was performed using restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments. Two nasal swab cultures were taken within 2 weeks before the operation. Subjects were classified as Staphylococcus aureus carriers if at least one culture was positive while those patients with both cultures found to be negative were classified as non-carriers. RESULTS: The prevalence of nasal S. aureus carriage in the final cohort was 22.1% (66/299), while no MRSA was detected in our study group. No significant differences in the TLR2 polymorphisms were observed between the study and the control groups. No associations were found between TLR2 haplotypes and the covariates of age, sex, NYHA, weight, height, BMI, CAD, smoking status and ESlog score. No differences were found between carriers and noncarriers regarding the allelic distribution of the TLR2 T-16934A SNP. Almost 93% of the patients who were screened for the presence of the TLR2 Arg753Gln (rs5743708) were GG wild type homozygous. Twenty one subjects from the study group (7.1%) were GA heterozygous, while no patient in either group was homozygous for the TLR2 Arg753Gln (rs5743708) mutation. TLR2 Arg753Gln genotyping showed that GA heterozygous patients were detected more frequently in the group of Staphylococcus aureus nasal carriers than in non-carrier adults. CONCLUSION: Our results suggest that the carrier status for the GA variant of the TLR2 Arg753Gln (rs5743708) polymorphism may be a risk factor for Staphylococcus aureus carriage.

Association Between RET (rs1800860) and GFRA1 (rs45568534, rs8192663, rs181595401, rs7090693, and rs2694770) Variants and Kidney Size in Healthy Newborns.

BACKGROUND: Abnormal congenital nephron number has been implicated in the pathogenesis of hypertension and renal disease. The RET receptor complex propagates signals essential for nephrogenesis and the RET c.1296G>A polymorphism, leading to aberrant splicing of exon 7, is associated with reduced kidney volume, a surrogate for nephron endowment. The glial cell-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFRA1) is a component of the RET receptor complex, and three alternatively spliced GFRA1 transcripts (with or without exon 5) have been identified. In rats, exclusion of exon 5 results in stronger GDNF binding affinity and RET activation. The aims of this study were to investigate further the relationship between RET c.1296G>A and kidney volume, and also to investigate the association between the GFRA1 polymorphisms near and within the alternatively spliced exon 5, as well as the functional 5'-UTR c.-193C>G with kidney volume. MATERIALS AND METHODS: The study included 188 healthy full-term newborns. Genotyping of the RET (NM_020975.4:c.1296G>A, rs1800860) and GFRA1 (NM_005264.5:c.-193C>G, rs45568534; c.419-87A>G, rs8192663; c.429G>A, rs181595401; c.433+127A>G, rs7090693; c.433+245A>G, rs2694770) polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism, minisequencing, or sequencing. Total kidney volume (TKV) was determined by ultrasound and normalized to body surface area (TKV/BSA). Both marker-by-marker and haplotype-based methods were used to test for associations between polymorphisms and TKV/BSA. RESULTS: TKV/BSA in RET c.1296A allele carriers was significantly lower compared with GG homozygotes (103 ± 23 vs. 110 ± 19 mL/m2, p = 0.034). c.429G>A was invariant in our sample. There was no association between any of the GFRA1 polymorphisms and renal volume. CONCLUSIONS: RET c.1296A may be a common susceptibility allele for nephron underdosing-related diseases. The 5'-UTR and intronic variants near exon 5 of GFRA1 are not associated with nephron endowment.

Epistatic interaction between common AGT G(-6)A (rs5051) and AGTR1 A1166C (rs5186) variants contributes to variation in kidney size at birth.

Low nephron number has been recognised as an important cardiovascular risk factor and recently a strong correlation between renal mass and nephron number has been demonstrated in newborns. The aim of this study was to investigate individual, as well as combined, effects of common variants of genes which encode for major components of the renin-angiotensin system (REN G10601A, AGT G(-6)A, ACE I/D, AGTR1 A1166C) on kidney size in healthy, full-term newborns. A significant additive main effect of the ACE I/D polymorphism, as well as an additive-by-additive interaction between AGT G(-6)A and AGTR1 A1166C variants, were found. The variance attributed to the epistatic effect was 27.9 ml(2)/m(4), which accounted for 73.8% of the interaction variance (37.8 ml(2)/m(4)), 66.4% of the genetic variance (42.0 ml(2)/m(4)) and 4.4% to the total phenotypic variance (628 ml(2)/m(4)). No other statistically significant main or epistatic effects were detected. Our results highlight the importance of considering gene-gene interactions as part of the genetic architecture of congenital nephron number, even when the loci do not show significant single locus effects. Unravelling the genetic determinants of low nephron number, along with early molecular screening, may well help to identify children at risk for cardiovascular disease.

Retrospective mutational analysis of NPHS1, NPHS2, WT1 and LAMB2 in children with steroid-resistant focal segmental glomerulosclerosis - a single-centre experience.

The aim of our study was to examine NPHS1, NPHS2, WT1 and LAMB2 mutations, previously reported in two thirds of patients with nephrotic syndrome with onset before the age of one year old. Genomic DNA samples from Polish children (n=33) with Steroid-Resistant Nephrotic Syndrome (SRNS) due to focal segmental glomerulosclerosis (FSGS), manifesting before the age of 13 years old, underwent retrospective analysis of NPHS1, NPHS2, WT1 (exons 8, 9 and adjacent exon/intron boundaries) and LAMB2. No pathogenic NPHS1 or LAMB2 mutations were found in our FSGS cohort. SRNS-causing mutations of NPHS2 and WT1 were detected in 7 of 33 patients (21%), including those with nephrotic syndrome manifesting before one year old: five of seven patients. Four patients had homozygous c.413G>A (p.Arg138Gln) NPHS2 mutations; one subject was homozygous for c.868G>A (p.Val290Met) NPHS2. A phenotypic female had C>T transition at position +4 of the WT1 intron 9 (c.1432+4C>T) splice-donor site, and another phenotypic female was heterozygous for G>A transition at position +5 (c.1432+5G>A). Genotyping revealed a female genotypic gender (46, XX) for the first subject and male (46, XY) for the latter. In addition, one patient was heterozygous for c.104dup (p.Arg36Profs*34) NPHS2; two patients carried a c.686G>A (p.Arg229Gln) NPHS2 non-neutral variant. Results indicate possible clustering of causative NPHS2 mutations in FSGS-proven SRNS with onset before age one year old, and provide additional evidence that patients with childhood steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis should first undergo analysis of NPHS2 coding sequence and WT1 exons 8 and 9 and surrounding exon/intron boundary sequences, followed by gender genotyping.