BACKGROUND Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One of the mechanisms used by malignant cells to escape immune surveillance is genetic or epigenetic modifications of tumor suppressor genes. Studies in B-cells and our previous report on the correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate tumor suppressor gene. Together, BACH2 and PRDM1 are described as having a fundamental role in oncogenesis, differentiation and apoptosis control of B and T lymphocytes during oxidative stress. Furthermore, several studies in mice model suggested that BACH2 gene is sensitive to DNA damage during aging and plays a role in the cellular senescence process. The impact of these genes in the immune response of the older adult is unclear. OUR HYPOTHESIS 1. Recent reports (including our previous study) on the transcription factor BACH2 and PRDM1 have highlighted their impact on immunological changes. It has been shown that BACH2 is highly sensitive to transcription-blocking DNA lesions in aged mice. Despite the growing interest in BACH's role, there have been many studies in mice models, studies on BACH2 in humans are still limited. This concept prompted us to investigate the variation of BACH2 and PRDM1 expression with age in major lymphocyte subsets from healthy individuals. 2. Apoptosis and senescence are two types of cellular response affected by cancer and aging processes, albeit through different mechanisms. Carcinogenesis is associated with a progressive reduction in the ability of the cells to trigger apoptosis and senescence. The lymphocytes apoptosis process and the markers of cell senescence were thus studied in both populations. 3. Chronic lymphocytic leukemia, one of the aging-related diseases, is characterized by the accumulation of “anergic” phenotypes in both B-cells and T-cells. In chronic lymphocytic leukemia, malignant cell anergy is associated with failure of inducing PRDM1 (BLIMP1), a critical regulator of differentiation into plasma cells, and that epigenetic modification account for such failure. We thus examined the expression of specific transcription factors BACH2 and PRDM1 in the leukemic lymphocytes subsets and compared the results with age-matched normal population. POPULATION & METHODS Blood samples were obtained from 60 healthy donors, aged from 20 to 90 years, and 41 untreated chronic lymphocytic leukemia were studied. Lymphocyte subsets (CD19+ B-cells, CD8 T-cells, CD4 T-cells, CD4 naïve T-cells, CD4 effector memory T-cells) were isolated for subsequent molecular analyses using the MACS Technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. BACH2, PRDM1, IL-4, IFNG, TP53, CDKN2A (p16INK4A), PDCD1 (PD-1) and CD274 (PD-L1) mRNA transcripts were quantified using qRT-PCR. BACH2 protein expression was examined by Western blotting. To investigate BACH2-target genes in apoptosis, we performed a “Human Apoptosis EpiTect ChIP qPCR Array” profiles including 84 key genes involved in programmed cell death. Etoposide was used to induce intracellular oxidative stress followed by cell apoptosis. Apoptotic cells were identified with the annexin-V-FITC apoptosis detection kit (BD Pharmingen). To measure ß-galactosidase by flow cytometry, we used Fluorescein-di-beta-D-galactopyranoside as a substrate for beta-galactosidase (Molecular Probes).RESULTS 1. BACH2 and PRDM1 gene expression were strongly correlated with age in healthy donor’s major lymphocyte subsets. BACH2 mRNA expression declined by age, with statistically significant differences observed in all lymphocyte subsets: CD4+, CD4+ naïve, CD4+ memory, CD8+ T-cells and CD19+ B-cells (p<0.0001, <0.0001, 0.0118, <0.0001 and 0.0004 respectively). In contrast, PRDM1 gene expression significantly increased with age in the same subpopulations. The phenotypic changes of immune lymphocytes are related to the BACH2 and PRDM1 gene expression. Along with the decline of BACH2, the older group had a severe decrease in number and quality of cytotoxic T-cells (p=0.0005 and <0.0001), while increasing the rate exhausted T-cells that highly expressed of PD-1 and PD-L1. Both PCCD1 (PD-1) and CD274 (PD-L1) gene expression had inversely correlated with BACH2 in the same lymphocyte subsets (p=0.0342; 0.0254 and 0.0429 respectively). Western blotting analysis demonstrated that BACH2 protein expressions in the T and B cell subpopulations significantly correlated with transcript expression. 2. Age-related BACH2 down-regulation enhanced the increasing of senescent cells and the resistance to apoptosis in the major lymphocyte subsets. We, further, found the correlation of BACH2 with senescence markers, such as p16INK4A (CDKN2A) and SA-β-GAL along with loss of CD28+ on T-lymphocytes. ChIP-qPCR Array demonstrated that BACH2 bound directly several genes involved in programmed cell death. A reduction in apoptosis was observed with age in CD4+ T-cells and CD19+ B-cells (p=0.03 in both subpopulations). As recently reported for BACH2-deficient mice, our data show that BACH2 down-regulation is strongly correlated with a decrease in CD4+ T-cell and CD19+ B-cell apoptosis (r=0.57 and 0.61). 3. Decreases in BACH2 mRNA expression were greater in CD4+ and CD8+ T-cells, as well as leukemic B-cell subpopulations from untreated CLL patients, compared with age-matched healthy donors (p=0.0006; 0.0003 and 0.0083, respectively). As expected, PRDM1 was significantly upregulated in the CD4+ and CD8+ T-cell subpopulations (p=0.003 and 0.001, respectively) from CLL patients but not their leukemic B-cells. CONCLUSION In this research work, we examined BACH2 and PRDM1 gene expression to investigate its potential as a predictive marker of aging. Our data suggest BACH2 and PRDM1 mRNA expression, in the inverse correlation, associated with the phenotypic changes of immune lymphocytes in aging. Age-related BACH2 down-regulation enhanced the increasing of exhausted phenotypes, senescent cells and resistance to cell apoptosis. BACH2 and PRDM1 could involve in cellular and immunological functions deterioration of the immunosenescence process. Further investigations on BACH2 and PRDM1 genetic mutations provide a better understanding of the role of the unbalanced expression of those genes in lymphocytes functions and survival according to age. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

This thesis presents genetic studies aiming at enlarging our knowledge regarding the genetic factors underlying two immune-mediated diseases, hypothyroidism and autoimmune Addison’s disease (AAD), in dogs and humans, respectively. Genetic and environmental factors are indicated to contribute to canine hypothyroidism, which can be considered a model for human Hashimoto’s thyroiditis (HT). In Paper I we performed the first genome-wide association (GWA) study of this disease in three high-risk dog breeds (Gordon Setter, Hovawart and Rhodesian Ridgeback). Using an integrated GWA and meta-analysis strategy, we identified a novel hypothyroidism risk haplotype located on chromosome 12 being shared by the three breeds. The identified haplotype, harboring three genes previously not associated with hypothyroidism, is independent of the dog leukocyte antigen region and significantly enriched across the affected dogs. In Paper II we performed a GWA study in another high-risk breed (Giant Schnauzer) and detected an associated locus located on chromosome 11 and conferring protection to hypothyroidism. After whole genome resequencing of a subset of samples with key haplotypes, we fine mapped the region of association that was subsequently screened for the presence of structural variants. We detected a putative copy number variant overlapping with the upstream region of the IFNA7 gene, which is located in a region of high genomic complexity. Remarkably, perturbed activities of type I Interferons have been extensively associated with HT and general autoimmunity. In Paper III we performed the first large-scale genetic study of human AAD, a rare autoimmune disorder characterized by dysfunction and ultimately destruction of the adrenal cortex. We resequenced 1853 immune-related genes comprising of their coding sequences, untranslated regions, as well as conserved intronic and intergenic regions in extensively characterized AAD patients and control samples, all collected in Sweden. We identified BACH2 gene as a novel risk locus associated with AAD, and we showed its independent association with isolated AAD. In addition, we confirmed the previously established AAD association with the human leukocyte antigen complex. The results of these studies will hopefully help increasing the understanding of such diseases in dogs and humans, eventually promoting their well-being.

Background: Type of 1 diabetes (T1D) is an autoimmune disease in which the cells of immune system attack the β-cells of pancreas. Consequently, destroyed β-cells do not produce insulin to reduce blood sugar levels. This disease is very complex, the pathogenesis is contributed by both genetic factors and environmental factors. In recent years, the number of individuals with T1D is increasing worldwide. Aims: The aim of this thesis was to investigate whether it is possible to predict T1D based on the expression profile of BACH2, CDC20, IGLL3P, EIF3A and TXNDC5 genes , which are involved in the development of immune system cells and insulin production. Another aim was to compare the expression of selected genes in children, in which the first detection of the disease may be done, and adults who suffer from prolonged T1D. The final goal was to compare the expression of individual selected genes in the HLA risk alleles DR04, DR03, DQA*05:01 a DQB*03:02. Methods: The DNA and RNA of patients with T1D and healthy individuals was isolated from blood. DNA was used to HLA genotyped. Isolated RNA was reverse transcribed into cDNA and then used in real-time PCR to determine the relative levels of gene expression. Conclusion: Significant results were obtained when the expression of BACH2, CDC20 and TXNDC5 genes...