Periodic Reporting for period 4 - DIALOY (Mosaic loss of chromosome Y (LOY) in blood cells - a new biomarker for risk of cancer and Alzheimer’s disease in men)
Periodo di rendicontazione: 2020-09-01 al 2021-02-28
Men in the entire world lives on average shorter lives compared with women and results delivered by the DIALOY project suggest that LOY in blood leukocytes is one of the underlying causes for this sex bias. Mosaic loss of chromosome Y (LOY) is a during lifetime acquired mutation occurring when the Y chromosome have been lost from a percentage of the peripheral blood cells. LOY is the most common human mutation and about 10%, 20% and more than 50% of men in the ages of 70, 80 and 90 years of age, are affected. Recent results suggest that men with LOY in blood might have an increased risk for various common disease and death. LOY in blood leukocytes can therefore help explain why men in the entire world lives shorter lives compared to females. In addition to age, genetic background, smoking and other exposures are known risk factors for LOY in blood. A central question is how can LOY in blood cells could increase the risk for disease in other organs and tissues? A likely explanation is that vital functions of immune cells in blood are compromised when affected with LOY, which is studied in the project. Furthermore, an overall aim of the project was to explore and establish LOY as a new predictive biomarker for cancer and Alzheimer’s disease in men. To this end, the following three objectives has been pursued:
• Objective 1 is to expand the studies of LOY as a biomarker by analyses of additional cohorts.
• Objective 2 is to investigate functional consequences of LOY through studies of changes of gene expression as a consequence of LOY and application of cell-sorting to understand the phenotypic effects from LOY in different cell-types in peripheral blood.
• Objective 3 is to develop improved methodology for LOY-detection with better sensitivity and specificity, compared to present methods, to promote the clinical utility of LOY.
• Objective 1 is to expand the studies of LOY as a biomarker by analyses of additional cohorts.
• Objective 2 is to investigate functional consequences of LOY through studies of changes of gene expression as a consequence of LOY and application of cell-sorting to understand the phenotypic effects from LOY in different cell-types in peripheral blood.
• Objective 3 is to develop improved methodology for LOY-detection with better sensitivity and specificity, compared to present methods, to promote the clinical utility of LOY.
During the project period, we and others have published new disease associations showing clear links between mosaic Y loss in blood and increased risk for morbidity and mortality in affected men. Notably, we published a new association with LOY and risk for Alzheimer’s disease and several papers established increased risk for different types of cancer, diabetes, age-related macular degeneration, to mention few. Importantly, our results first published in 2014 (showing an increased risk for death overall) has been reproduced and validated.
An important task in the DIALOY project was to take these epidemiological results further, and study if men diagnosed with different types of disease were affected with LOY in different types of leukocytes, i.e. circulating immune cells in blood. This would indicate a direct effect from LOY on different types of disease. To this end, we collected and analyzed LOY in six major types of leukocytes in men diagnosed with prostate cancer and Alzheimer's disease and controls. Remarkably, our results (accepted for publication 2021) show that men with Alzheimer’s disease were primarily affect with LOY in NK-cells while men with prostate cancer displayed an increased level of LOY in T-cells and granulocytes. In another published paper in the project, we showed that the frequency of leukocytes with LOY typically increase in frequency over time within the blood of serially studied men. These results show similarities between LOY and the process of clonal hematopoiesis; a result that will be further studied in future projects.
Another goal of the DIALOY project was to investigate functional consequences of LOY in immune cells of blood. To this end, we have been studying changes in gene expression as a consequence of LOY, both in different cell types sorted from blood by FACS and RNAseq, as well as by single-cell transcriptomic analyses using the 10X Chromium system. Results from this project shows that leukocytes without the Y chromosome display a disturbed expression of about 500 autosomal genes important for various biological functions, including genes important for immune checkpoint regulation and other normal immune functions such as immune surveillance. These results (accepted for publication 2021) suggest that LOY might have negative effects on vital immune system functions, connected with accelerated pathological processes in the entire body and increased risk for mortal disease. Hence, our results reinforce the hypothesis that LOY could play a direct physiological role in the etiology of all disease with strong immune component. In addition, we describe in a paper published in Nature 2019 that LOY shares a genetic predisposition with cancer susceptibility, diabetes as well as reproductive ageing in women. The shared risk variants for LOY and disease highlight genes involved in cell cycle regulation, DNA damage response, genomic imbalance and apoptosis. These results help explain why some men are affected by LOY while others are not, results that are complementary to our previous finding showing that age and smoking are important risk factors. Thus, the overlap in genetic predisposition for LOY and disease suggest that LOY could be a barometer of genomic instability and disease risk in other organs and it is likely that such predisposition acts in parallel with a direct physiological effect from LOY in leukocytes.
We also investigated and developed new and improved methodology for LOY-detection with increased sensitivity and specificity to promote the clinical utility of LOY testing. First, we assisted in the development of a custom SNP-array for best practice LOY-analyses, a product that is now on the market and used by us and other research groups. Furthermore, we developed a sensitive ddPCR protocol that can be used in research for robust and quick LOY-assessment and validation. We have also worked on developing a novel method for LOY detection using cell surface markers. This would enable rapid and sensitive LOY analysis with improved clinical utility. Preliminary results from a successful pilot project is currently prepared for publication.
An important task in the DIALOY project was to take these epidemiological results further, and study if men diagnosed with different types of disease were affected with LOY in different types of leukocytes, i.e. circulating immune cells in blood. This would indicate a direct effect from LOY on different types of disease. To this end, we collected and analyzed LOY in six major types of leukocytes in men diagnosed with prostate cancer and Alzheimer's disease and controls. Remarkably, our results (accepted for publication 2021) show that men with Alzheimer’s disease were primarily affect with LOY in NK-cells while men with prostate cancer displayed an increased level of LOY in T-cells and granulocytes. In another published paper in the project, we showed that the frequency of leukocytes with LOY typically increase in frequency over time within the blood of serially studied men. These results show similarities between LOY and the process of clonal hematopoiesis; a result that will be further studied in future projects.
Another goal of the DIALOY project was to investigate functional consequences of LOY in immune cells of blood. To this end, we have been studying changes in gene expression as a consequence of LOY, both in different cell types sorted from blood by FACS and RNAseq, as well as by single-cell transcriptomic analyses using the 10X Chromium system. Results from this project shows that leukocytes without the Y chromosome display a disturbed expression of about 500 autosomal genes important for various biological functions, including genes important for immune checkpoint regulation and other normal immune functions such as immune surveillance. These results (accepted for publication 2021) suggest that LOY might have negative effects on vital immune system functions, connected with accelerated pathological processes in the entire body and increased risk for mortal disease. Hence, our results reinforce the hypothesis that LOY could play a direct physiological role in the etiology of all disease with strong immune component. In addition, we describe in a paper published in Nature 2019 that LOY shares a genetic predisposition with cancer susceptibility, diabetes as well as reproductive ageing in women. The shared risk variants for LOY and disease highlight genes involved in cell cycle regulation, DNA damage response, genomic imbalance and apoptosis. These results help explain why some men are affected by LOY while others are not, results that are complementary to our previous finding showing that age and smoking are important risk factors. Thus, the overlap in genetic predisposition for LOY and disease suggest that LOY could be a barometer of genomic instability and disease risk in other organs and it is likely that such predisposition acts in parallel with a direct physiological effect from LOY in leukocytes.
We also investigated and developed new and improved methodology for LOY-detection with increased sensitivity and specificity to promote the clinical utility of LOY testing. First, we assisted in the development of a custom SNP-array for best practice LOY-analyses, a product that is now on the market and used by us and other research groups. Furthermore, we developed a sensitive ddPCR protocol that can be used in research for robust and quick LOY-assessment and validation. We have also worked on developing a novel method for LOY detection using cell surface markers. This would enable rapid and sensitive LOY analysis with improved clinical utility. Preliminary results from a successful pilot project is currently prepared for publication.
We are world leading in the field of LOY analyses in single-cell transcriptomic data sets (scRNAseq). With this new method, which is gaining huge popularity in the field of human genetics, we can study gene expression in single-cells and thus, the effects of LOY at the most relevant and high resolution level. We are continuously generating and analyzing data sets using scRNAseq and similar approaches which will give us the opportunity to understand in great details what actually happens in cells after losing the Y chromosome. The information gained from these analyses is also used to identify targets for improved LOY-detection and novel approaches for clinical settings. Still today, most such analyses are based on DNA level technologies, but we envision that both RNA and protein level markers can be utilized for this purpose. A long term goal of the ongoing LOY project is a the translational benefit from our studies, by implementing LOY-testing in general populations as a mean to identify men with increased risk for various disease. LOY could also be useful for clinical applications such as improved diagnoses and improved treatment strategies (such as immunotherapies). In the end, we are confident that our research will lead to improved health of men and possibly; change the fact that men in the entire world lives on average shorter lives compared with women.