8^th International Conference on Human Genetics and Genetic Diseases November 25-26, 2019 Madrid, Spain

Biography:

Ben Borokhovsky is a current MD candidate student at Cooper Medical School of Rowan University. He completed his undergraduate education at Widener University and graduated Summa Cum Laude with a degree in Biochemistry. He did research throughout his entire undergraduate education and has consistently been asked to speak at Widener’s Research Symposium and has even given a keynote presentation on his senior thesis, for which he won a first place award. During his time at Widener University, Ben’s research involved looking at gene regulation of specific SR proteins and the roles they play in lipid metabolism using an alternative splicing model in D. melanogaster to better understand molecular and genetic links to obesity in humans.

Abstract:

The method of gene regulation underlying lipid storage related to obesity is poorly understood, yet alternative splicing (AS) appears to be an important mechanism for proper lipid storage. CPT1 (carnitine palmitoyltransferase 1) is a beta-oxidation enzyme involved in the breakdown of fatty acids. The gene coding for CPT1 is alternatively spliced by the SR protein 9G8 to produce two different products that vary in their activity. My project was to investigate if there was a difference in the AS phenotype among flies with decreased expression of 9G8, the SR protein antagonist RSF1, and the SR protein transporter TRN. First, a quantitative PCR (qPCR) protocol needed to be developed to detect the isoforms of CPT1. cDNA was generated from wild type flies that were fed (0 hrs), starved (65 hrs) or refed to optimize the likelihood that both isoforms would be produced. Following a published protocol, a qPCR procedure was carried out that skipped the elongation step (73) and instead cycled from the standard annealing temperature (60) to the denaturation step (95 ). Additionally, the PCR plasticware resulted in technical difficulties and required troubleshooting. Even though there were difficulties, the qPCR reactions saw good results. The preps were good quality confirmed by qPCR by having rp49 gene come in at 15-17 cycles. The individual isoforms 6A and 6B were both detected at distinct cycle numbers showing that the isoform ratios change depending on which gene KD is occurring and would directly affect lipid storage and contribute to fat reserves. Future directions include running triglyceride (TG) assays to determine if there is a change in TG levels among the 9G8, RSF1, and TRN knockdown flies and then investigate if their splicing patterns changed as well.

Biography:

Litvinova Larisa has completed her MD and currently she is working as Head of Laboratory, Immunology and Cell Biotechnology, Immanuel Kant Baltic Federal University, Kaliningrad, Russian Federation. She has published more than 180 papers in reputed journals.

Abstract:

Introduction: Visfatin has multidirectional effects-pro-inflammatory, stimulating the production of pro-inflammatory cytokines, and neuro-, angioprotective properties. Adipokine has an insulin-mimetic effect and stimulates insulin production. In this regard, the aim of the study was to study the role of visfatin in the development of type 2 diabetes in patients with obesity.

Methodology & Theoretical Orientation: Serum glucose levels were determined using a biochemistry analyzer. Using the method of flow fluorimetry, the content of visfatin, insulin and leptin in the blood plasma was determined using commercial test systems; gene expression level - by PCR.

Findings: In the group of obese patients with type 2 diabetes, the content of leptin, insulin and glucose in the blood plasma was higher, which was the case for patients without type 2 diabetes and control (p <0.05). The level of visfatin in blood plasma was higher than 802.10 (101.0 - 1145.12) pg / ml than in patients with obesity without type 2 diabetes 371.81 (40.83 - 513.58) pg / ml and below the control values (p <0.05). In the group of obese patients with type 2 diabetes, the level of visfatin positively correlated with the level of leptin (r = 0.513) and negatively with the level of insulin (r = -0.435) (p <0.05). However, in patients without type 2 diabetes, the level of visfatin had positive correlations with the content of insulin (r = 0.812) and leptin (r = 0.767) in blood plasma (p <0.05). The expression level of the NAMPT gene (visfatin) was increased in all obese patients with type 2 diabetes in all fat depots, compared with obese patients without it. In obese patients with type 2 diabetes, the level of NAMPT gene expression in subcutaneous adipose tissue was 15 times higher than the control group (p <0.05), while in patients without type 2 diabetes it did not differ from the control, which may indicate the formation of adipokine plasma levels, mainly due to its production of subcutaneous fat depot in patients with type 2 diabetes. The level of NAMPT gene expression in the greater omentum positively correlated with the level of Visfatin in blood plasma in the group of obese patients with type 2 diabetes (r = 0.5 p <0.05). The level of NAMPT gene expression in the mesentery of the small intestine negatively correlated with BMI in the group of obese patients with type 2 diabetes (r = 0.4 p <0.05).

Conclusion & Significance: Thus, visfatin can have different effects on the development of type 2 diabetes in obese patients, mediated by tissue-specific features of its secretion, as indicated by (1) the revealed formation of plasma levels of adipokine due to subcutaneous fat depot in patients with type 2 diabetes; (2) the detected protective effects of visfatin in patients without type 2 diabetes, due to the established positive relationship between the level of visfatin and plasma insulin production in this category of patients.

Biography:

Ms. Ainur Sibagatova is an aspiring researcher. She holds a master degree in health administration. Currently she works in the Gerontology center of Medical Centre Hospital of President’s Affairs Administration of the Republic of Kazakhstan. The interest of her research is the prevention of the age of associated diseases, mainly cardiovascular diseases.

Abstract:

Introduction: Cardiovascular Disease (CVD) is the leading cause of death in Kazakhstan. In the structure of CVD, hypertension takes the first place and leads to remodeling of the myocardium and carotid arteries, which significantly increases cardiovascular risk. A number of studies have been carried out that established the genetic characteristics associated with the risk of hypertension and target organ damage in hypertension, but not in Kazakh population.

The aim of this project is to study the molecular genetic predictors of remodeling of the cardiovascular system in HTN in Kazakh population.

Materials and methods: The sample size is 1000 people. There were 4 groups of 250 hypertensive individuals, age - up to 61 years.

1. Group without remodeling
2. with carotid artery remodeling (CR), intima-media complex (IMC) > 0.9 mm
3. with myocardial remodeling (MR) LVMI ≥115 in men, ≥95 in women, relative wall thickness ≥0.43
4. With remodeling of the myocardium and carotid artery.

Clinical, laboratory, instrumental data were collected. Biological samples were taken for genotyping for 120 SNPs known by association with a thickening of IMC or with left ventricular hypertrophy based on GWAS Catalog - EMBL-EBI and Varsome Clinical and PubMed publications. Genotyping was performed using QuantStudio 12K Flex Real-Time PCR System using array technology.

Preliminary Results: According to a preliminary analysis of 120 SNPs, three polymorphic variants (rs75330306, rs1879734, rs12465515 (p <0.05)) are associated with MR, because of a significant association in groups with MR and in the group RM+CR. rs1879734 and rs12465515 were previously associated with mitral valve prolapse in the European population, and rs75330306 was previously associated with Idiopathic dilated cardiomyopathy in the African American ancestry.

Biography:

Ewa Hordyjewska-Kowalczyk is a young scientist focusing her interest on human genetics and the development of limbs. She has been studying mutations linked to monogenic diseases (Cleidocranial Dysplasia, IFAP syndrome) as well as a complex disorder – Clubfoot.

Abstract:

Introduction: Cleidocranial Dysplasia (CCD) is an autosomal dominant disorder occurring 1 in a million births. The most common phenotypic manifestation of CCD includes hypoplastic or absent clavicles, failure of cranial suture closure and dental anomalies. These defects were linked to mutations in the RUNX2 (Runt-related transcription factor 2), a master regulator of osteoblast differentiation, cartilage, and bone development. Although the gene is known, novel variants are constantly being discovered. The purpose of the study was the functional characterization of six mutations found in Polish patients with CCD.

Methodology & Theoretical Orientation: Members of 11 Polish families were genotyped for variants in the RUNX2 gene. Then to assess the pathogenicity of 6 variants functional studies were carried out, including tests for transactivation potential of RUNX2 mutants and subcellular localization.

Findings: All of the found variants are present in the Runt domain of RUNX2 and are associated with the presence of supernumerary teeth among all patients. What is more, three variants are newly reported missense changes. Furthermore, we showed a significant decrease in the RUNX2 transactivation activity of all mutants, and although only two variants are within Nuclear Localization Signal (NLS), and other two affect the putative NLS, all investigated mutants localize to the cytoplasm.

Conclusion & Significance: The report brings functional evidence on the pathogenicity of found mutations which have a detrimental effect on the RUNX2 and trigger the CCD phenotype. These data not only increase our knowledge about the function of RUNX2 domains but also add to the clinical description and the phenotype/genotype correlation in the patients with these mutations.

Biography:

Abstract:

Introduction: The functioning of mitochondria is disrupted and is associated with a decrease in ATP synthesis and organelle division during obesity under conditions of oxidative stress and increased production of ROS. The aim of the study was to evaluate the expression of genes associated with mitochondrial division / fusion processes in obese patients with type 2 diabetes.

Methodology & Theoretical Orientation: The expression level of genes was determined by real-time PCR. Calculations of the level of relative expression of the studied genes and the significance of differences were determined relative to the control group in the REST program.

Findings: An increase in the level of gene expression of the pro-inflammatory transcription factor NF-kB (2 times relatively healthy donors, p <0.05) in patients with obesity with type 2 diabetes in liver biopsy specimens showed inflammation in the liver parenchyma, whereas in patients without type 2 diabetes of this type, on the contrary, was lower (100 times, p <0.05) of control values. In obese patients with type 2 diabetes, the level of TFAM gene expression increased by 1.3 times (p <0.05), and in obese patients without it, it did not change relative to the control. In patients with obesity with type 2 diabetes, the expression level of the MFN2 and DRP1 gene did not change, and in patients with obesity without it, it decreased by 8.7 times and 100 times, respectively, relative to the control (p <0.05). In this study, in patients with obesity without type 2 diabetes, a decrease in the expression of genes characterizing mitochondrial division and fusion — DRP1 and MFN2 — was recorded; accordingly, a deficiency of proteins involved in mitochondrial fusion may indicate a decrease in cellular respiration and metabolism.

Conclusion & Significance: Thus, an increase in the level of TFAM gene expression in obese patients with type 2 diabetes mellitus indicates an increase in mtDNA transcription, while mitochondrial dysfunction mediated by an increase in the expression level of mitochondrial transcription factor A (TFAM), in the presence of an inflammatory process in the liver in obese patients, may be a factor contributing to the formation of type 2 diabetes.

Biography:

Michał Kiełbus completed his Ph.D. thesis in 2016, was focused on finding the prognostic factors in brain cancer. As a post-doc he partially remained in the cancer field and his current project investigates the molecular basis of Epithelial-Mesenchymal Transition (EMT). He acquired experience in designing and conducting all the steps of well-planned functional experiments, starting from virtual cloning, genome editing and ending with different assays including e.g. cell imaging or gene expression assessment. His particular interests are, exploring the molecular mechanisms that change cell phenotype and result in the induction of a disease process.

Abstract:

Epithelial-mesenchymal transition (EMT) and, its inverse process, mesenchymal-epithelial transition (MET), are physiological processes that also play important roles in disease. Mounting evidence correlates EMT with the poor outcome of multiple neoplasias, as well as metastasis and drug resistance. Thus, the molecular mechanisms that regulate EMT are an important area of cancer research and drug targeting. In order to study EMT, different exogenous reporter systems that can signal phenotypical changes are commonly used. Nonetheless, the use of exogenous reporters that are unable to describe molecular events, including the interference of alternative promoters, regulatory elements or epigenetic mechanisms that modulate promoter activation, making some data unreliable. Trying to overcome those limitations, we developed and validated a reporter cell line by modifying the endogenous VIMENTIN gene (a gene associated with EMT) using gene editing CRISPR/Cas9 technology. Specifically, we successfully knocked-in a DNA sequence, containing a self-cleaving peptide followed by a fluorescent protein in-frame with VIMENTIN into the genome of H2170 lung cancer cells. Our data uniquely illustrate that the Vimentin reporter cells are a reliable model for studying EMT and MET. The Vimentin reporter cells allow spatio-temporal observation of cellular plasticity with respect to their mesenchymal/epithelial in vitro and in the future in vivo. This is an excellent model to study the molecular mechanisms of EMT/MET as well as a robust platform to screen for new anti-cancer drugs.

Biography:

Maria Vulf, PhD is a researcher at the Center for Immunology and Cellular Biotechnology, Immanuel Kant Baltic Federal University, Kaliningrad. She has published about 20 papers in reputed journals.

Abstract:

Introduction: Abdominal obesity and type 2 diabetes mellitus (T2DM) occupy one of the leading positions among the factors contributing to the reduction of working capacity, disability and mortality. The system of heat shock proteins (HSP) is a universal and powerful mechanism for protecting cells from damage of various origins. The aim of this work was to identify the features of the expression of heat shock proteins (HSP70) in obese patients.

Methodology: The material for the study was biopsies of the liver, adipose tissue (omentum, mesentery, subcutaneous adipose tissue) and peripheral blood mononuclear cells. The study included 54 participants. The study group consisted of 26 obese patients with T2DM, 11 patients without carbohydrate metabolism disorders and comparison group of 17 donors without changing the parameters of carbohydrate and fat metabolism. To determine the levels of gene expression, the method was RT-PCR using qPCRmix-HS SYBR reagents (Eurogen). The morphological and functional state of the liver was diagnosed by histological method.

Findings: The indicators of carbohydrate metabolism in patients with T2DM were increased, relative to patients without T2DM and a comparison group. Histological analysis revealed changes in the violation of lipid metabolism in the liver, despite the absence of clinically significant changes. In obese patients in all groups were diagnosed with non-alcoholic fatty liver disease. Scientists suggest that the induction of heat shock proteins protects against insulin resistance against obesity, but the basic mechanisms are still not understood. The level of HSP70 expression in all types of adipose tissue and liver biopsy samples turned out to be not statistically significant, while in patients with peripheral blood mononuclear cells a significant increase of 80 times was recorded.

Conclusion: Thus, in the future we plan to increase the patient sample, evaluate the production of this protein in the studied tissues, which will reveal the role of HSP70 in obese patients.

Biography:

Khaziakhmatova Olga, PhD, is a researcher at the Center for Immunology and Cellular Biotechnology, IKBFU. The main areas of work are cell technology and she also participates in stem cell and immunocompetent cell research projects.

Abstract:

Introduction: Currently, traumatology actively uses ceramic materials with calcium phosphate coating (CP). Such materials have a stimulating effect on multipotent mesenchymal stromal cells (MMSCs) and immunocompetent cells. So, their use involves an assessment of the impact of these materials on the development of tumor cells.

Methodology: The Jurkat 5332 cell line and human adipose-derived MMSCs (AMMSCs) were examined. 3D culture was simulated by adding to the cell culture the substrates from commercially pure titanium with rough (Ra=2-5 µm) CP microarc coating. The cultures were used: 2D with Jurkat T cells (JTCs) on plastic surface; 2D co-culture of JTCs and AMMSCs on plastics; 3D with JTCs and CP matrix; 3D with JTCs, AMMSCs, and CP matrix.

Findings & Conclusion: Both 2D and 3D JTC cultures showed an increase in the CD45RO receptor expression that led to increasing number of CD45RO+CD45RA+ cells. Probably, JTCs restored the partial maturation and differentiation. Vice versa, the expression of CD45RO receptor and the number of CD45RO+CD45RA+ cells decreased in case of JTCs and AMMSCs co-cultivation. A similar reaction of the cells was revealed in the 3D culture of JTCs and AMMSCs. Thus, JTCs with AMMSCs co-cultivation may promote the preservation of the tumor cell phenotype and survival. An additional confirmation of the differentiation of JTCs was a decrease in the number of CD25 + and CD95 + cells in the presence of matrices. Moreover, decreased content of IFN-γ and IP-10 in cell supernatants. The cultivation of AMMSC and Jurkat revealed a significant increase in the production of IFN-γ (3 times) and IP-10 (10 times). But despite this, the level of chemokine IP-10 in the presence of matrices with CP coating was significantly reduced; the content of INF-γ remained practically unchanged. Cultivation of JTCs with matrices with CP coating resulted in a 3-fold decrease in the level of MCP-1 in culture supernatants. The cultivation of AMMSC with Jurkat in a 2D model revealed an increase in the concentration of chemokines MCP-1 (20 times) and RANTES (400 times). In the 3D cultivation model, the secretory activity of the mixed culture decreased and was similar to the level of AMMSC 3D monoculture. 

Biography:

Komar Aleksandra is a biologist at the Center for Immunology and Cellular Biotechnology (Kaliningrad, Russian Federation). She studied cell technology in a cell laboratory in 2018 (Vilnius, Lithuania). She studied genetic and biochemical methods for a year of study in postgraduate school. This work is part of future PhD thesis.

Abstract:

Introduction: The anti-inflammatory cytokine interleukin-10 (IL10) may play a protective role in type 2 diabetes (T2D). The Wnt5a signaling pathway regulates the production of a wide range of cytokines in stellate cells of the liver, so we suggest a relationship between the expression of the WNT5a gene in the liver and the content of IL-10 in the circulation in obese patients with and without T2D.

Methodology & Theoretical Orientation: Serum glucose levels were determined on a biochemical analyzer; the concentration of IL10 in blood plasma - by flow fluorimetry; WNT5a gene expression level - by PCR; quantitative content of protein WNT5A – by Western Blotting.

Findings: Expression of the gene Wnt5a in the group of obese patients without diabetes increased by 1.7 times (p<0.05); the group of obese patients with diabetes increased by 1.47 times (p<0.05). Liver protein Wnt5a levels consistent with gene expression Wnt5a. Plasma blood IL10 levels in patients with type 2 diabetes were lower than in patients without type 2 diabetes [1]. Pereira C. et al.  was found the inhibitory effect of Wnt5a overexpression on the anti-inflammatory cytokine IL10 in a culture of isolated macrophages and a decreased in the release of pro-inflammatory cytokines [1]. While a low IL10 concentration in blood plasma can contribute to the development of chronic inflammation in patients with T2D [2]. The level of IL10 negatively correlated with the level of expression of the WNT5a gene in the liver in patients with type 2 diabetes (r=-0,548, Ñ€<0.05). High expression of WNT5A in the liver was associated with a high IL10 plasma level in patients with obesity without type 2 diabetes and a low IL10 plasma level in patients with obesity with type 2 diabetes.

Conclusion & Significance: Thus, a high level of WNT5a gene expression can be associated with the protective effect of IL 10 in obese patients without T2D.

Biography:

Abstract:

Introduction: The steady growth of patients with type 2 diabetes mellitus (T2D) in the Republic of Kazakhstan over the past decade indicates the need to develop a program for the prevention of T2D. A change in the expression of candidate genes can lead to impaired insulin synthesis, a decrease in the number of insulin receptors, β-cell dysfunction, and obesity. The purpose of this study is to analyze the association of the SNPs with biochemical and anthropometric parameters of T2D in representatives of the Kazakh population.

Materials and methods: In total, 139 patients with T2D and 100 randomly selected patients with no signs of disease were registered in the current study. Cases of T2D are diagnosed based on World Health Organization criteria. The samples were ethnically homogeneous and included only the Kazakhs. The biochemical (total cholesterol, LDL, HDL, glucose and HOMA-IR index) and anthropometric indicators were evaluated.

Statistical processing of the results was carried out using the software package: MS Office Excel 2013, STATISTICA v. 6.0. Differences were considered significant when p ≤ 0.05.

Results: Two of the tested loci (rs7901695 and rs7903146 in the TCF7L2 gene) showed statistically significant associations with T2D. The TCF7L2 gene is involved in the processes of adipogenesis, differentiation of adipose tissue, and in the regulation of pancreatic β-cells development and functioning. A statistically significant relationship was found between rs7901695 and rs7903146 in the TCF7L2 gene and indicators of glucose metabolism, lipid spectrum and BMI. Insulin resistance is a major factor in the development of T2D. The study found associations of hyperglycemia, high levels of LDL, hypertriglyceridemia and a decrease in HDL with genetic markers rs2237892 in the KCNQ1, rs7756992 and rs7754840 gene in the CDKAL1 gene in patients with T2D. The fact that these genes are active in β-cells confirms the notion that β-cell dysfunction is fundamental in the pathogenesis of T2D.

These results suggest that in the Kazakh population, these polymorphisms are associated with an increased risk of developing T2D through β-cell dysfunction, regardless of obesity. Thus, based on the results, it can be concluded that in the Kazakh population, the leading role is played by polymorphisms that affect the level of synthesis and secretion of insulin in pancreatic β-cells.

Biography:

Abstract:

Introduction: The increasing prevalence of type 2 diabetes (T2D) underlines the urgent need for proactive strategies to prevent and control T2D. A fairly large number of studies show that T2D is a complex metabolic disease caused by lifestyle, environment and genetic factors.

The aim of this study was to search for genetic markers associated with the development of T2D in individuals of the Kazakh population.

Materials and methods: The study included 139 patients with type 2 diabetes. The established WHO diagnostic criteria were used to diagnose T2D. The control group was a random sample of 100 patients with no signs of T2D. The samples were ethnically homogeneous and consisted of Kazakh individuals. The genotyping was performed on a new generation QuantStudio 12K Flex instrument, Life Technologies.Statistical analysis was performed using the Statistica for Windows 7.0 software (StatSoft, USA).

Results: Of the 25 previously tested SNPs, we identified eight (rs17584499, rs7903146, rs7756992, rs7754840, rs 2237892, rs4712524 (P <0.001), rs1333051 and rs7901695 (P <0.01)) statistically significant SNPs. In addition, seven of the tested SNPs (rs 2237896, rs 2237897, rs2383208, rs1575972 rs4402960, rs1470579, rs163184) showed a nominal connection with T2D, (OR) from 1.7 - 2.6, (P <0.05), which suggests that these options could potentially be used in the Kazakh population for prognostic testing of T2D. It should be specially noted that some of the studied polymorphic genetic markers (rs10460009, rs6583826, rs9295474, rs8181588, rs3888647, rs8050136, rs4712523, rs3773506, rs1049549, rs11642841) did not demonstrate a connection with T2D. The odds ratio varied between 0.7-2.93, CI (0.41-1.19 to 0.77-5.71). In summary, the results of this study represent a preliminary understanding of T2D pathogenesis in Kazakhs, what helps can improve preventative measures to reduce its risk.

Biography:

Seidalin Nazar is an oncologist. He has extensive clinical experience. Also, he conducts research, in particular the study of a genetic predisposition to the development of cancer, since it provides an understanding of the potential mechanisms of tumor development.

Abstract:

Introduction: The incidence of prostate cancer (PCa) due to increased life expectancy is steadily increasing. Currently, a list of polymorphisms associated with the risk of tumors, their course and treatment response has been determined, but there is no convincing evidence for their clinical use. Objective is to study the frequency of occurrence of polymorphisms in healthy men and patients with prostate cancer in the Kazakh population.

Materials and methods: Two groups of Kazakh - patients with prostate cancer (n = 480) and control (n = 479) were recruited during 2017-2019. DNA from blood samples were genotyped with 120 SNPs chip using qPCR (QuantStudio 12K, ThermoFisher). Genotype annotation was performed with ThermoFisher cloud.

Findings: The main and control groups are comparable in age. The average time from the diagnosis of prostate cancer to the time of inclusion in the study was 3.1 years, with a median of 2.7 years. The stage of the disease at the time of diagnosis in the main group of men with prostate cancer was: stage I - 12.6%; stage II - 40.9%; stage III - 30.8%; stage IV - 15.7%. The minimum allele frequency (MAF) of rs10187424 SNP for the control group was 39.5% (C-allele), while in the group with PCa, the C-allele was major (64.3%). Distribution by genotypes in the control group C / C - 6.2%, C / T - 66.7% and T / T - 27.1%. In the group with prostate cancer, C / S - 55%, C / T - 18.6% and T / T - 26.4%. After Bonferroni correction, p values were obtained using the log-additive inheritance model: LO p value <0.001 (4.761574e-23).

Conclusion: Minor allele (C- allele) оf rs10187424 polymorphism located on 2 chromosome in VAMP8 gene has strong association with prostate cancer in Kazakh populaton. It should be noted that MAF in Kazakhs is very close to East Asians (38,6%) and South Asians (35.5%).

Biography:

Darta Pupola has finished University of Latvia as a biologist. She has experience in genomics, cell culturing and signaling pathway analysis in a field of oncology and GIT microbiome, where combining all knowledge from different kind of fields it becomes possible to look at disease and health conditions as a complex interaction network.

Abstract:

Introduction: H. pylori infections are present in 80% of Latvian population thus increasing the susceptibility of numerous of the gastric tract diseases, including gastric adenocarcinoma.¹ The 1^st line H. pylori eradication therapy includes treatment with clarithromycin in combination with amoxicillin or metronidazole and a proton pump inhibitor. However, potential adverse events caused by such therapies to microbiome are insufficiently studied. Therefore, the aim of this study was to evaluate the long-term effects of H. pylori eradication on gastrointestinal (GIT) microbiome.

Methodology & Theoretical Orientation: The assessment of H. pylori eradication therapy on GIT microbiome was performed on 120 faecal samples that were acquired from 60 adults: samples from each individual were collected before starting the eradication therapy, and one year after the final treatment. Samples were collected in OC-Sensor (Eiken Chemical Co., Tokyo, Japan) sample collection containers and stored at -86°C. Total DNA was extracted using FastDNA Spin Kit for Soil (MP Biomedicals, USA) and was followed by 16S rRNA V3 gene sequencing employing Ion Torrent Personal Genome Machine (Life Technologies, USA). The obtained raw 16S reads were analyzed using QIIME v.1.9.0 and UPARSE v.7.0.1001.

Conclusion & Significance: Overall microbiome community composition remained stable between pre- and post-eradication microbiome samples, however, shifts between predominant enterotypes as well as positive correlation for certain bacteria between the two categories was found in relation to age, individual, experience respiratory and/or allergic diseases and if the eradication therapy was used as prescribed. Modest global differences at the community level exist between individuals before and after the eradication therapy when considering the long-term impact; however, the microbiome structure is more related with the patient-specific parameters, such as age or experienced diseases, rather than by the eradication therapy itself.

Biography:

Mª Luisa Villahermosa is the Director of the R&D Department at GENOMICA, with 17 years´ experience in developing innovative molecular diagnostic products in oncology and microbiology areas, from concept to Industry. She is creative and has an excellent understanding and product development know-how. The NGS technology was one of the innovation techniques that were introduced in the Department to offer services and to use it internally during the development and validation processes.

Abstract:

Formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the Next Generation Sequencing (NGS) analysis. The efficiency of Exome sequencing from FFPE tissue depends of the amount and quality of DNA extracted. To generate accurate NGS data, we have implemented a specific protocol in order to obtain accurate sequencing of the exomes. We analyzed 21 tissue samples obtained at diagnosis from patients with solid tumors using Ion ampliseq^TM Exome RDY (Thermofisher) with the objective of detecting germinal variants. Genomic DNA (gDNA) concentration was quantified using Qubit^® fluorometry, and its quality was determined using the RNase P Taqman. Correct NGS libraries were obtained considering the degradation status: we increase the quantity of gDNA for performing the library according with the data obtained with the RNase P analysis in each sample.

We obtained successful NGS results in 90.5% of cases: -15 samples showed Optimal results (Uniformity in sequence coverage >91.26%; Target base coverage at 20x >94%), being the quantity of gDNA 2-10ng/µl in 6 samples, and 10-50ng/µl in 9 samples; these samples showed optimal quality of gDNA. -5 samples showed Medium results (Uniformity in sequence coverage 50-80%; Target base coverage at 20x 60-85%), being the quantity of gDNA in all samples 2-5ng/µl, and the quality of the gDNA was mostly medium-low. -1 sample showed Low result (Uniformity in sequence coverage: 40%; Target base coverage at 20x 50%) being the quantity of gDNA 5 ng/µl, and the quality was very close to the medium values.

We have implemented an additional step before the Library generation, which allow us to increase the quantity of gDNA according with the obtained quantity/quality values. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.