Improved branch and bound algorithm for detecting SNP-SNP interactions in breast cancer

BACKGROUND: Single nucleotide polymorphisms (SNPs) in genes derived from distinct pathways are associated with a breast cancer risk. Identifying possible SNP-SNP interactions in genome-wide case–control studies is an important task when investigating genetic factors that influence common complex traits; the effects of SNP-SNP interaction need to be characterized. Furthermore, observations of the complex interplay (interactions) between SNPs for high-dimensional combinations are still computationally and methodologically challenging. An improved branch and bound algorithm with feature selection (IBBFS) is introduced to identify SNP combinations with a maximal difference of allele frequencies between the case and control groups in breast cancer, i.e., the high/low risk combinations of SNPs. RESULTS: A total of 220 real case and 334 real control breast cancer data are used to test IBBFS and identify significant SNP combinations. We used the odds ratio (OR) as a quantitative measure to estimate the associated cancer risk of multiple SNP combinations to identify the complex biological relationships underlying the progression of breast cancer, i.e., the most likely SNP combinations. Experimental results show the estimated odds ratio of the best SNP combination with genotypes is significantly smaller than 1 (between 0.165 and 0.657) for specific SNP combinations of the tested SNPs in the low risk groups. In the high risk groups, predicted SNP combinations with genotypes are significantly greater than 1 (between 2.384 and 6.167) for specific SNP combinations of the tested SNPs. CONCLUSIONS: This study proposes an effective high-speed method to analyze SNP-SNP interactions in breast cancer association studies. A number of important SNPs are found to be significant for the high/low risk group. They can thus be considered a potential predictor for breast cancer association

Double-bottom chaotic map particle swarm optimization based on chi-square test to determine gene-gene interactions

Gene-gene interaction studies focus on the investigation of the association between the single nucleotide polymorphisms (SNPs) of genes for disease susceptibility. Statistical methods are widely used to search for a good model of gene-gene interaction for disease analysis, and the previously determined models have successfully explained the effects between SNPs and diseases. However, the huge numbers of potential combinations of SNP genotypes limit the use of statistical methods for analysing high-order interaction, and finding an available high-order model of gene-gene interaction remains a challenge. In this study, an improved particle swarm optimization with double-bottom chaotic maps (DBM-PSO) was applied to assist statistical methods in the analysis of associated variations to disease susceptibility. A big data set was simulated using the published genotype frequencies of 26 SNPs amongst eight genes for breast cancer. Results showed that the proposed DBM-PSO successfully determined two-to six-order models of genegene interaction for the risk association with breast cancer (odds ratio > 1.0; value < 0.05). Analysis results supported that the proposed DBM-PSO can identify good models and provide higher chi-square values than conventional PSO. This study indicates that DBM-PSO is a robust and precise algorithm for determination of gene-gene interaction models for breast cancer

The role of common genetic variants for predicting the modulation of cardiovascular outcomes

Attrition is a major issue in the drug development process with 79% of clinical failures due to safety and efficacy concerns. Genetic research can provide supporting evidence of a clear causal relationship between the drug target and disease or reveal unintended effects through associations with non-relevant phenotypes informing on potential drug safety. However, due to the underlying genetic architecture, it is often unclear which gene or variant in the loci identified through genetic analyses is driving the association. Due to recent advancements in CRISPR-Cas9 gene-editing, it is now possible to relatively easily perform whole gene knock-out studies and single base-edits to validate genetic findings of the most likely causal variant and gene. Utilising a combination of genetic approaches and functional studies can provide supporting evidence of the therapeutic profile and potential effects of drug therapies and improve our overall understanding of biological pathways and disease mechanisms. The primary aim of this thesis is to provide genetic data to support the ongoing clinical development of hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitors (PHIs) for treating anaemia of chronic kidney disease (CKD). Genome-wide association studies (GWAS) were used to identify genetic variants lying within or nearby genes encoding the drug target (prolyl hydroxylase [PHD] enzymes). These identified variants were used in Mendelian Randomisation analysis and phenome-wide association studies to genetically mirror the pharmaceutical effects of PHIs and investigate cardiovascular safety. Functional validation studies were employed to functionally validate a genetic variant for use as a proxy and to obtain a better understanding of the downstream causal pathways and biological mechanisms of the drug target. In summary, this thesis demonstrates how a combination of genetic analyses and functional validation studies is a powerful approach to validate GWAS results and further characterise therapeutic effects. This PhD project identified relevant genetic markers to genetically proxy therapeutic modulation of biomarker levels through PHD inhibition and could potentially inform further research using patient-level clinical data from Phase III trials

Defining the molecular, genetic and transcriptomic mechanisms underlying the variation in glycation gap between individuals

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.The discrepancy between HbA1c and fructosamine estimations in the assessment of glycaemia has frequently been observed and is referred to as the glycation gap (G-gap). This could be explained by the higher activity of the fructosamine-3-kinase (FN3K) deglycating enzyme in the negative G-gap group (patients with lower than predicted HbA1c for their mean glycaemia) as compared to the positive G-gap group. This G-gap is linked with differences in complications in patients with diabetes and this potentially happens because of dissimilarities in deglycation. The difference in deglycation rate in turn leads to altered production of advanced glycation end products (AGEs). These AGEs are both receptor dependent and receptor independent. It was hypothesised that variations in the level of the deglycating enzyme fructosamine-3-kinase (FN3K) might be as a result of known Single Nucleotide Polymorphisms (SNPs): rs1056534, rs3848403 and rs1046896 in FN3K gene, SNP in ferroportin1/SLC40A1 gene (rs11568350 linked with FN3K activity), differentially expressed genes (DEGs), differentially expressed transcripts or alternatively spliced transcript variants. Previous studies reported accelerated telomere length shortening in patients with diabetes. In this study, 184 patients with diabetes were included as dichotomised groups with either a strongly negative or positive G-gap. This study was conducted to analyse the differences in genotype frequency of specific SNPs via real time qPCR,determine soluble receptors for AGE (sRAGE) concentration via ELISA, finding association of sRAGE concentration with SNPs genotype, and evaluate relative average telomere length ratio via real time qPCR. This study also aimed at the investigation of underlying mechanisms of G-gap via transcriptome study for the identification of the DEGs and differentially expressed transcripts and to consequently identify pathways, biological processes and diseases linked to situations in which DEGs were enriched. The relative length of the telomere was normalised to the expression of a single copy gene (S). Chi-squared test was used for estimating the expected genotype frequencies in diabetic patients with negative and positive G-gap. Genotype frequencies of FN3K SNPs (rs1056534, rs3848403 and rs1046896) and SLC40A1/ferroportin1 SNP (rs11568350) polymorphisms within the studied groups were non-significant. With respect to genotypes, the rs1046896 genotype (CT) and rs11568350 genotype (AC) were only found in heterozygous state in all the investigated cohorts. No association between sRAGE concentration and FN3K SNPs (rs3848403 and rs1056534) was observed as the sRAGE concentration was also found not to be different between the groups. Similarly, the relative average telomere length was not different in both groups. Plasma sRAGE levels were not different in the cohort studied even though the Wolverhampton Diabetes Research Group (WDRG) previously reported that AGE is higher in positive G-gap. The latter is a more likely consequence of lower FN3K activities. In this study, it was found that SNPs in the FN3K/ferroportin1 gene are not responsible for the discrepancy in average glycaemia. The transcriptomic study via RNA-Seq mapped a total of 64451 gene transcripts to the human transcriptome. The DEGs and differentially expressed transcripts were 103 and 342 respectively (p 1.5). Of 103 DEGs, 61 were downregulated in G-gap positive and 42 were upregulated in positive G-gap individuals while 14 genes produced alternatively spliced transcript variants. Four pathways (Viral carcinogenesis, Ribosome, Phagosome and Dorso-ventral axis) were identified in the bioinformatics analysis of samples in which DEGs were enriched. These DEGs were also found to be associated with raised blood pressure and glycated haemoglobin (conditions that coexist with diabetes). Future analysis based on these results will be necessary to elucidate the significant drivers of gene expression leading to the G-gap in these patients

Abstracts from the 50th European Society of Human Genetics Conference: Posters

International audienc

Training Manual ICAR Short course on Application of advanced molecular methods in marine fishery resource management, conservation and sustainable mariculture

Molecular Biology and Biotechnology has undergone incredible progress in this decade mainly due to the rapid advancements in DNA sequencing technologies. Marine biology and fishery science also reaped the fruits of these modern inventions improving our understanding regarding complex adaptations in aquatic organisms. Fish Genetics have evolved into genomics incorporating knowledge about neutral and non-neutral markers. A project called Genome 10k was started by the international community of scientists for sequencing the genome of 10000 vertebrates. Whole genomes of many marine organisms are now available which provided insights into the evolution of many important traits. Transcriptome sequencing provides insights into expressed genes and metagenome sequencing provides information regarding the microbes present in environment. All these technologies are rapid and cost effective. Over years, these technologies provided exciting opportunities for understanding ecology and evolution. Genomic information can also be sustainably utilized to enhance productivity of mariculture activities by selective breeding, genetic improvement and manipulation of economically important traits. ICAR-Central Marine Fisheries Research Institute has contributed significantly to marine biotechnology research in the country and played a pivotal role in development of marine fisheries sector. The short course on “Application of advanced molecular methods in marine fisheries resource management, conservation and sustainable mariculture” conducted in ICAR-CMFRI from 24th October, 2018 to 2nd November, 2018 is specially designed to provide exposure to various applications of molecular tools in fisheries resource management, conservation of biodiversity and mariculture. I hope this compendium of lectures and protocols will be extremely useful for the participants to effectively utilize the knowledge in their own area of research. Simultaneously, on behalf of ICAR-CMFRI, I warmly welcome all the participants from various institutions and wish them all success in their future endeavors. I am sure that this training will result in new knowledge, collaborations and friendships

New developments on the pathogenesis of systemic lupus erythemstosus

Tese de doutoramento, Medicina (Pediatria), Universidade de Lisboa, Faculdade de Medicina, 2018Systemic Lupus Erythematosus (SLE) is a challenging autoimmune disease, with a complex etiopathogenesis and an unpredictable clinical course. In a large cohort of juvenile-onset SLE patients, we found that major infections were common, were associated with active disease and its treatment, and resulted in noteworthy morbidity. New biomarkers to guide the judicious use of immunosuppressive drugs and new treatment strategies with fewer side effects would, therefore, have an enormous impact in the management of these patients. In order to reach these goals we used the modern tools of molecular biology and focused on two of the most important complications of SLE: lupus nephritis and macrophage activation syndrome. Firstly, we identified the kidney lupus nephritis specific microRNA (miRNA) signature, which reflected mainly cell proliferation. MiRNAs are noncoding RNAs responsible for post-transcriptional gene silencing. These key regulatory molecules control the expression of multiple genes, so its dysregulation can contribute to sustained pathology. We showed that miR-26a and miR-30b were significantly decreased in the kidneys and urine of lupus nephritis patients. In vitro, the knockdown of miR-26a and miR-30b caused the proliferation of human mesangial cells and increased the expression of genes related to the cell cycle, including CCNE2, E2F8, MAD2L1, MYBL1 and POLQ. The Human Epidermal Growth Factor Receptor 2 (HER2) is a protein previously known to regulate miR-26a and miR-30b expression. Trastuzumab, a monoclonal antibody against HER2, used in breast cancer treatment, produces therapeutic actions precisely by up-regulating miR-26a and miR-30b. In human mesangial cells we also found that trastuzumab increases these two miRNAs. We hypothesized that HER2 also played a role in the pathogenesis of lupus nephritis and indeed we identified a dramatic overexpression of HER2 in the glomeruli and tubular compartments of the kidneys of lupus nephritis patients. The same pattern was not seen in the kidneys of healthy individuals or in other proliferative glomerulonephritides, including post-streptococcus glomerulonephritis, IgA nephropathy and granulomatosis with polyangiitis. Furthermore, in the lupus-prone NZM2410 mice we identified a highly increased expression of HER2, which correlated with disease activity. Finally, we showed that urinary HER2 was significantly increased in lupus nephritis and that its levels increased during flares, were higher in class III and class IV lupus nephritis and correlated with urinary proteincreatinine and monocyte chemoattractant protein 1 (MCP1) and vascular cell adhesion protein 1 (VCAM1) levels. We, therefore, established a strong rationale to use trastuzumab to block HER2 and decrease cell proliferation and damage in lupus nephritis. Regarding the macrophage activation syndrome, we were interested in the characterization of hemophagocytes. These are activated macrophages that have engulfed other hematopoietic cells. Traditionally they have been associated with the development of cytopenias in several life-threatening cytokine storm syndromes. New data have challenged this concept, since pancytopenia occurs in the absence of hemophagocytosis in mice and, in humans, over 40% of patients with macrophage activation syndrome do not have hemophagocytes in bone marrow aspirates. On the other hand, subclinical hemophagocytosis is detected in more than 50% of patients with systemic juvenile idiopathic arthritis, but only 10% develop a life-threatening macrophage activation syndrome. Thus the function and significance of hemophagocytes remained mysterious. Recent evidence demonstrated that environmental factors, particularly the cytokine milieu, determine the macrophage activation status in a continuum ranging from M1 to M2. M1 macrophages, driven by interferon γ, typically acquire proinflammatory properties and are associated with tissue damage, whereas M2 macrophages have more heterogeneous stimuli and functions, being associated with immunoregulation, tissue remodeling and fibrosis. Since interferon γ is the hallmark cytokine of hemophagocytic lymphohistiocytosis, one would expect to find that hemophagocytes express M1 surface receptors. We characterized the transcriptional phenotype of mouse Toll-like receptor 9 (TLR9) – induced hemophagocytes and described the surface phenotype of human bone marrow hemophagocytes. Interestingly, murine hemophagocytes had up-regulation of genes associated with the M2 and not the M1 phenotype. Immunohistochemical analyses in bone marrow samples from a uniquely diverse cohort of patients with hemophagocytic syndromes showed universal staining of hemophagocytes for the M2 marker CD163, but rarely for CD206 or CD64. Collectively, these data support the hypothesis that hemophagocytes have immunoregulatory functions and open new doors for the study of the pathogenesis of this syndrome. Finally, we were interested in characterizing the transcriptome of SLE, by RNAsequencing, in order to better understand the mechanisms that are responsible for chronicity. Monocytes from SLE patients exhibited a globally dysregulated gene expression. The transcriptome was not simply altered by the activation of a set of genes, but was also qualitatively different. Splicing patterns and polyadenylation were significantly altered and SLE monocytes expressed novel transcripts, an effect that was replicated by exposing control monocytes to lipopolysaccharide (LPS). We further identified increased circulating endotoxin in SLE patients, suggesting that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism. In conclusion, with these different projects, which globally focused on the transcriptome and epigenome of SLE, we identified novel pathways and challenged the current paradigms. We described new mechanisms of disease, including the role of LPS in transforming qualitatively the SLE transcriptome and a putative immunoregulatory function for hemophagocytes. We demonstrated that miR-26a, miR-30b and HER2 control cell proliferation in lupus nephritis and that are promising biomarkers. Most importantly, our work rose the possibility of using anti-HER2 drugs for lupus nephritis management, opening the door to a new treatment strategy in this disease.Fundação Calouste Gulbenkian, Fundação Champalimau