Transcriptional Changes in Regulatory T Cells From Patients With Autoimmune Polyendocrine Syndrome Type 1 Suggest Functional Impairment of Lipid Metabolism and Gut Homing

Autoimmune polyendocrine syndrome type I (APS-1) is a monogenic model disorder of organ-specific autoimmunity caused by mutations in the Autoimmune regulator (AIRE) gene. AIRE facilitates the expression of organ-specific transcripts in the thymus, which is essential for efficient removal of dangerous self-reacting T cells and for inducing regulatory T cells (Tregs). Although reduced numbers and function of Tregs have been reported in APS-I patients, the impact of AIRE deficiency on gene expression in these cells is unknown. Here, we report for the first time on global transcriptional patterns of isolated Tregs from APS-1 patients compared to healthy subjects. Overall, we found few differences between the groups, although deviant expression was observed for the genes TMEM39B, SKIDA1, TLN2, GPR15, FASN, BCAR1, HLA-DQA1, HLA-DQB1, HLA-DRA, GPSM3 and AKR1C3. Of significant interest, the consistent downregulation of GPR15 may indicate failure of Treg gut homing which could be of relevance for the gastrointestinal manifestations commonly seen in APS-1. Upregulated FASN expression in APS-1 Tregs points to increased metabolic activity suggesting a putative link to faulty Treg function. Functional studies are needed to determine the significance of these findings for the immunopathogenesis of APS-1 and for Treg immunobiology in general.publishedVersio

BRCA1 Norway: comparison of classifcation for BRCA1 germline variants detected in families with suspected hereditary breast and ovarian cancer between different laboratories

Pathogenic germline variants in Breast cancer susceptibility gene 1 (BRCA1) predispose carriers to hereditary breast and ovarian cancer (HBOC). Through genetic testing of patients with suspected HBOC an increasing number of novel BRCA1 variants are discovered. This creates a growing need to determine the clinical significance of these variants through correct classification (class 1–5) according to established guidelines. Here we present a joint collection of all BRCA1 variants of class 2–5 detected in the four diagnostic genetic laboratories in Norway. The overall objective of the study was to generate an overview of all BRCA1 variants in Norway and unveil potential discrepancies in variant interpretation between the hospitals, serving as a quality control at the national level. For a subset of variants, we also assessed the change in classification over a ten-year period with increasing information available. In total, 463 unique BRCA1 variants were detected. Of the 126 variants found in more than one hospital, 70% were interpreted identically, while 30% were not. The differences in interpretation were mainly by one class (class 2/3 or 4/5), except for one larger discrepancy (class 3/5) which could affect the clinical management of patients. After a series of digital meetings between the participating laboratories to disclose the cause of disagreement for all conflicting variants, the discrepancy rate was reduced to 10%. This illustrates that variant interpretation needs to be updated regularly, and that data sharing and improved national inter-laboratory collaboration greatly improves the variant classification and hence increases the accuracy of cancer risk assessment.publishedVersio

Sensing foul AIRE: Investigating possible reporter genes for AIRE mutations

The autoimmune regulator protein, known as AIRE, is a potent transcriptional regulator active in medullary thymic epithelial cells (mTECs) of the thymus, where it is able to switch on the expression of thousands of genes commonly only expressed in specialised peripheral tissues. This ability of AIRE makes it a crucial component in the immune system, specifically for the process of negative selection, in which T-cells are evaluated in their ability to recognise the body’s own proteins. This works as a check-point to avoid autoimmunity, and T-cells that bind to AIRE induced proteins are terminated as they are considered dangerous for the organism. Disruption of AIRE function by mutations leads to the disease autoimmune polyendocrine syndrome type 1 (APS-1), in which autoimmune T-cells initiate destructive processes affecting a variety of functions in the body. Clinically, APS-1 is defined as the presence of at least two out of three major manifestations: Addison’s disease (adrenal insufficiency), hypoparathyroidism and chronic mucocutaneous candidosis. However, patients may not necessarily present the major manifestations, and may also exhibit a variety of other manifestations, both of which may be related to the severity of the underlying mutation. AIRE consists of a number of functional domains; a CARD sequence used for AIRE dimerisation, a SAND domain for general DNA interaction, and two PHD zinc fingers, one used for histone interaction, and the other for protein recruitment and interaction. AIRE induction works in a stochastic manner, targeting genes that are passively downregulated by methylated histone marks, or that are otherwise actively repressed. Therefore AIRE induced genes differ between cells because of the sheer amount of inducible genes, as well as between different cell types, because different cell types will repress different genes. In order to study AIRE function and inform larger sequencing and GWAS studies, we are aiming to develop a functional screening assay for AIRE mutations, using a deep mutational scanning approach. This would attempt to characterise the functional effect of any hypothetical mutation within AIRE and would require a robust reporter gene. This would be a gene with high expression in AIRE wildtype, but low expression in an AIRE mutant, while preferably encoding a cell surface protein for easier FACS sorting. To identify these reporter genes we aimed to develop a robust cell system with AIRE. This cell system needed to be amenable to large-scale transfection and FACS sorting, and be robustly expressing functional AIRE proteins. We, therefore, investigated the expression of known AIRE reporter genes, curated from the literature, and evaluated the usability of these genes as possible reporters. Furthermore, we developed a protocol for AIRE inducible gene discovery using RNA sequencing and evaluated different methodological approaches to this. We successfully established a robust cell system based on AIRE transfected HEK293FT cells, which exhibited substantial AIRE expression. By using qPCR probes for known AIRE induced genes, we also confirmed that AIRE was functionally active. We found that the previously reported AIRE regulated genes KRT14 and S100A8 could be used as reporter genes based on lower expression in selected AIRE mutants. We found that RNAseq is highly consistent between experiments and across methodological approaches when it comes to library preparation, and correlates well with results using qPCR. However, we were unable to identify new reporter genes fitting our criteria, and the reporter gene candidates KRT14 and S100A8 were too weakly expressed to be detected by RNAseq. Comparing the AIRE wildtype with untransfected cells yielded substantial transcriptome differences, consistent with the literature, yet did not yield usable reporter genes. Comparing the AIRE mutants R257X and C311Y with the wildtype, in order to find downregulated genes in the mutants, we found a large population of upregulated genes in the R257X mutant and little difference between C311Y and wildtype. Neither of these mutants has previously been investigated using transcriptome analysis, and so it is uncertain how representative these results are, but they are consistent across our experiments. Western blot analysis showed some degradation in all transfected populations, yet substantial degradation of the C311Y mutant, suggesting a possible instability in this variant. AIRE is a fascinating transcriptional regulator able to induce the expression of repressed genes, but the knowledge of AIRE and its function is still incomplete. The failure of our RNAseq approach to detect AIRE reporter genes indicates that changes in methodology are required. Such changes may still render a deep mutational scanning approach a viable option for the purpose of studying AIRE

Transcriptional Changes in Regulatory T Cells From Patients With Autoimmune Polyendocrine Syndrome Type 1 Suggest Functional Impairment of Lipid Metabolism and Gut Homing

Autoimmune polyendocrine syndrome type I (APS-1) is a monogenic model disorder of organ-specific autoimmunity caused by mutations in the Autoimmune regulator (AIRE) gene. AIRE facilitates the expression of organ-specific transcripts in the thymus, which is essential for efficient removal of dangerous self-reacting T cells and for inducing regulatory T cells (Tregs). Although reduced numbers and function of Tregs have been reported in APS-I patients, the impact of AIRE deficiency on gene expression in these cells is unknown. Here, we report for the first time on global transcriptional patterns of isolated Tregs from APS-1 patients compared to healthy subjects. Overall, we found few differences between the groups, although deviant expression was observed for the genes TMEM39B, SKIDA1, TLN2, GPR15, FASN, BCAR1, HLA-DQA1, HLA-DQB1, HLA-DRA, GPSM3 and AKR1C3. Of significant interest, the consistent downregulation of GPR15 may indicate failure of Treg gut homing which could be of relevance for the gastrointestinal manifestations commonly seen in APS-1. Upregulated FASN expression in APS-1 Tregs points to increased metabolic activity suggesting a putative link to faulty Treg function. Functional studies are needed to determine the significance of these findings for the immunopathogenesis of APS-1 and for Treg immunobiology in general

BRCA1 Norway: comparison of classifcation for BRCA1 germline variants detected in families with suspected hereditary breast and ovarian cancer between different laboratories

Pathogenic germline variants in Breast cancer susceptibility gene 1 (BRCA1) predispose carriers to hereditary breast and ovarian cancer (HBOC). Through genetic testing of patients with suspected HBOC an increasing number of novel BRCA1 variants are discovered. This creates a growing need to determine the clinical significance of these variants through correct classification (class 1–5) according to established guidelines. Here we present a joint collection of all BRCA1 variants of class 2–5 detected in the four diagnostic genetic laboratories in Norway. The overall objective of the study was to generate an overview of all BRCA1 variants in Norway and unveil potential discrepancies in variant interpretation between the hospitals, serving as a quality control at the national level. For a subset of variants, we also assessed the change in classification over a ten-year period with increasing information available. In total, 463 unique BRCA1 variants were detected. Of the 126 variants found in more than one hospital, 70% were interpreted identically, while 30% were not. The differences in interpretation were mainly by one class (class 2/3 or 4/5), except for one larger discrepancy (class 3/5) which could affect the clinical management of patients. After a series of digital meetings between the participating laboratories to disclose the cause of disagreement for all conflicting variants, the discrepancy rate was reduced to 10%. This illustrates that variant interpretation needs to be updated regularly, and that data sharing and improved national inter-laboratory collaboration greatly improves the variant classification and hence increases the accuracy of cancer risk assessment

GWAS for autoimmune Addisons disease identifies multiple risk loci and highlights AIRE in disease susceptibility

Autoimmune Addisons disease (AAD) is characterized by the autoimmune destruction of the adrenal cortex. Low prevalence and complex inheritance have long hindered successful genetic studies. We here report the first genome-wide association study on AAD, which identifies nine independent risk loci (P&amp;lt;5x10(-8)). In addition to loci implicated in lymphocyte function and development shared with other autoimmune diseases such as HLA, BACH2, PTPN22 and CTLA4, we associate two protein-coding alterations in Autoimmune Regulator (AIRE) with AAD. The strongest, p.R471C (rs74203920, OR=3.4 (2.7-4.3), P=9.0x10(-25)) introduces an additional cysteine residue in the zinc-finger motif of the second PHD domain of the AIRE protein. This unbiased elucidation of the genetic contribution to development of AAD points to the importance of central immunological tolerance, and explains 35-41% of heritability (h(2)). Autoimmune Addisons disease is a rare complex disease, which has not yet been characterized by non-biased genetic studies. Here, the authors perform the first GWAS for the disease, identifying nine loci including two coding variants in the gene Autoimmune Regulator (AIRE).Funding Agencies|Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) [sens2017513]; KG Jebsen Foundation; Research Council of NorwayResearch Council of Norway; Swedish Research CouncilSwedish Research CouncilEuropean Commission; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation; Health Authorities of Western Norway; Torsten and Ragnar Soderberg Foundations; Novo Nordisk FoundationNovo Nordisk Foundation; Swedish Society for Medical Research</p

GWAS for autoimmune Addison's disease identifies multiple risk loci and highlights AIRE in disease susceptibility

Autoimmune Addison’s disease (AAD) is characterized by the autoimmune destruction of the adrenal cortex. Low prevalence and complex inheritance have long hindered successful genetic studies. We here report the first genome-wide association study on AAD, which identifies nine independent risk loci (P < 5 × 10−8). In addition to loci implicated in lymphocyte function and development shared with other autoimmune diseases such as HLA, BACH2, PTPN22 and CTLA4, we associate two protein-coding alterations in Autoimmune Regulator (AIRE) with AAD. The strongest, p.R471C (rs74203920, OR = 3.4 (2.7–4.3), P = 9.0 × 10−25) introduces an additional cysteine residue in the zinc-finger motif of the second PHD domain of the AIRE protein. This unbiased elucidation of the genetic contribution to development of AAD points to the importance of central immunological tolerance, and explains 35–41% of heritability (h2)