Genetic variants regulating immune cell levels in health and disease.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

The longitudinal dynamics and natural history of clonal haematopoiesis.

Clonal expansions driven by somatic mutations become pervasive across human tissues with age, including in the haematopoietic system, where the phenomenon is termed clonal haematopoiesis1-4. The understanding of how and when clonal haematopoiesis develops, the factors that govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression remains limited5,6. Here we track 697 clonal haematopoiesis clones from 385 individuals 55 years of age or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A and TP53) to more than 50% per year (SRSF2P95H). Growth rates of clones with the same mutation differed by approximately ±5% per year, proportionately affecting slow drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole-genome sequences of haematopoietic colonies from 7 individuals from an older age group, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations drove expansion only later in life, whereas TET2-mutant clones emerged across all ages. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterize the lifelong natural history of clonal haematopoiesis and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.

GWAS of genetic factors affecting white blood cell morphological parameters in Sardinians uncovers influence of chromosome 11 innate immunity gene cluster on eosinophil morphology.

Few genome-wide association studies (GWAS) analyzing genetic regulation of morphological traits of white blood cells have been reported. We carried out a GWAS of 12 morphological traits in 869 individuals from the general population of Sardinia, Italy. These traits, included measures of cell volume, conductivity and light scatter in four white-cell populations (eosinophils, lymphocytes, monocytes, neutrophils). This analysis yielded seven statistically significant signals, four of which were novel (four novel, PRG2, P2RX3, two of CDK6). Five signals were replicated in the independent INTERVAL cohort of 11 822 individuals. The most interesting signal with large effect size on eosinophil scatter (P-value = 8.33 x 10-32, beta = -1.651, se = 0.1351) falls within the innate immunity cluster on chromosome 11, and is located in the PRG2 gene. Computational analyses revealed that a rare, Sardinian-specific PRG2:p.Ser148Pro mutation modifies PRG2 amino acid contacts and protein dynamics in a manner that could possibly explain the changes observed in eosinophil morphology. Our discoveries shed light on genetics of morphological traits. For the first time, we describe such large effect size on eosinophils morphology that is relatively frequent in Sardinian population.

Polygenic risk score and biochemical/environmental variables predict a low-risk profile of age-related macular degeneration in Sardinia.

PURPOSE: To ascertain the prevalence and clinical and genetic features of age-related macular degeneration (AMD) in subjects living in the Lanusei valley, Central Sardinia, Italy, involved in a study on ageing (SardiNIA project). METHODS: A total of 814 volunteers aged ≥ 50 years, randomly selected from the SardiNIA project dataset, were included. A color fundus (CF) photograph of the 30° central retina of each eye was obtained and graded according to the Age-Related Eye Disease Study system. Life-style choices were investigated using standardized questionnaires. The concentrations of several inflammatory biomarkers (i.e., complement component, fibrinogen, and C-reactive protein) were measured. Polygenic risk score (PRS) was calculated and compared with results obtained from a European cohort. RESULTS: A total of 756 subjects had gradable CF photographs for AMD detection. In 91.3%, no signs of AMD were observed. The prevalence rates of early and late AMDs were 6.9% and 0.6%, respectively. A total of 85% of subjects were physically active; only 13.5% were current smokers. Low concentrations of complement component, fibrinogen, and C-reactive protein were found. We calculated the polygenic risk scores (PRS) using 40 AMD markers distributed on several candidate genes in Europeans and Sardinians. The mean PRS value was significantly lower in Sardinians than in the Europeans (0.21 vs. 0.248, respectively, p = 1.18 × 10-77). CONCLUSIONS: In our cohort, most subjects showed no sign of any AMD type and late AMD was a condition rarely observed. Results of genetic, biochemical, and life-style investigation support the hypothesis that Sardinia population may present of a peculiar background with a protective effect against AMD development.

Autoimmunity Is a Significant Feature of Idiopathic Pulmonary Arterial Hypertension.

Rationale: Autoimmunity is believed to play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear whether this is causative or a bystander of disease and if it carries any prognostic or treatment significance. Objectives: To study autoimmunity in IPAH using a large cross-sectional cohort. Methods: Assessment of the circulating immune cell phenotype was undertaken using flow cytometry, and the profile of serum immunoglobulins was generated using a standardized multiplex array of 19 clinically validated autoantibodies in 473 cases and 946 control subjects. Additional glutathione S-transferase fusion array and ELISA data were used to identify a serum autoantibody to BMPR2 (bone morphogenetic protein receptor type 2). Clustering analyses and clinical correlations were used to determine associations between immunogenicity and clinical outcomes. Measurements and Main Results: Flow cytometric immune profiling demonstrates that IPAH is associated with an altered humoral immune response in addition to raised IgG3. Multiplexed autoantibodies were significantly raised in IPAH, and clustering demonstrated three distinct clusters: "high autoantibody," "low autoantibody," and a small "intermediate" cluster exhibiting high concentrations of ribonucleic protein complex. The high-autoantibody cluster had worse hemodynamics but improved survival. A small subset of patients demonstrated immunoglobulin reactivity to BMPR2. Conclusions: This study establishes aberrant immune regulation and presence of autoantibodies as key features in the profile of a significant proportion of patients with IPAH and is associated with clinical outcomes.

Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

Genetic variant within CDK6 regulates immune response to palbociclib treatment.

Everyone carries a set of genetic variants that contribute to regulation of the levels of blood cells, with unknown clinical impact. One of them, rs445 within the cell-cycle checkpoint gene CDK6, reduces the levels of myeloid cell types including granulocytes. We treated CD3+ T cells and whole blood with palbociclib in 41 individuals, who were stratified by genotype for analyses. In T cells we assessed cell cycle and apoptosis, whereas in whole blood, apoptosis in activated (CD11b+), unactivated (CD11b-) granulocytes, cytotoxic (CD8 + CD4-), and helper (CD8-CD4+) T cells. We find that rs445 modulates the immune response of CD8+ T cells. It also increases the level of apoptotic CD11b + activated granulocytes after palbociclib treatment, which, in synergy with neutropenia, may affect drug related adverse events. These results suggest that the effect of palbociclib treatment may depend on underlying genetically encoded individual immune response as well as the direct response to the drug.

Immunologic resilience and COVID-19 survival advantage.

BACKGROUND: The risk of severe coronavirus disease 2019 (COVID-19) varies significantly among persons of similar age and is higher in males. Age-independent, sex-biased differences in susceptibility to severe COVID-19 may be ascribable to deficits in a sexually dimorphic protective attribute that we termed immunologic resilience (IR). OBJECTIVE: We sought to examine whether deficits in IR that antedate or are induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection independently predict COVID-19 mortality. METHODS: IR levels were quantified with 2 novel metrics: immune health grades (IHG-I [best] to IHG-IV) to gauge CD8+ and CD4+ T-cell count equilibrium, and blood gene expression signatures. IR metrics were examined in a prospective COVID-19 cohort (n = 522); primary outcome was 30-day mortality. Associations of IR metrics with outcomes in non-COVID-19 cohorts (n = 13,461) provided the framework for linking pre-COVID-19 IR status to IR during COVID-19, as well as to COVID-19 outcomes. RESULTS: IHG-I, tracking high-grade equilibrium between CD8+ and CD4+ T-cell counts, was the most common grade (73%) among healthy adults, particularly in females. SARS-CoV-2 infection was associated with underrepresentation of IHG-I (21%) versus overrepresentation (77%) of IHG-II or IHG-IV, especially in males versus females (P < .01). Presentation with IHG-I was associated with 88% lower mortality, after controlling for age and sex; reduced risk of hospitalization and respiratory failure; lower plasma IL-6 levels; rapid clearance of nasopharyngeal SARS-CoV-2 burden; and gene expression signatures correlating with survival that signify immunocompetence and controlled inflammation. In non-COVID-19 cohorts, IR-preserving metrics were associated with resistance to progressive influenza or HIV infection, as well as lower 9-year mortality in the Framingham Heart Study, especially in females. CONCLUSIONS: Preservation of immunocompetence with controlled inflammation during antigenic challenges is a hallmark of IR and associates with longevity and AIDS resistance. Independent of age, a male-biased proclivity to degrade IR before and/or during SARS-CoV-2 infection predisposes to severe COVID-19.

Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function.

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.

PRF1 mutation alters immune system activation, inflammation, and risk of autoimmunity.

BACKGROUND: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines-cytokine storm. OBJECTIVE: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). METHODS: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. RESULTS: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10-4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10-5, OR = 0.82. CONCLUSION: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

CXCR3 Identifies Human Naive CD8+ T Cells with Enhanced Effector Differentiation Potential.

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

Kidney size in relation to ageing, gender, renal function, birthweight and chronic kidney disease risk factors in a general population.

BACKGROUND: The relationship of kidney size to ageing, kidney function and kidney disease risk factors is not fully understood. METHODS: Ultrasound length and parenchymal kidney volume were determined from a population-based sample of 3972 Sardinians (age range 18-100 years). We then identified the subset of 2256 'healthy' subjects to define age- and sex-specific reference ranges (2.5-97.5 percentile) of kidney volume. Logistic regression (accounting for family clustering) was used to identify the clinical characteristics associated with abnormally large kidneys or abnormally small kidneys. RESULTS: In the healthy subset, kidney volume and length increased up to the fourth to fifth decade of life followed by a progressive decrease in men, whereas there was a gradual kidney volume decrease throughout the lifespan of women. In the whole sample, independent predictors of lower kidney volume (<2.5 percentile for age and sex) were male sex, low body mass index, short height, low waist:hip ratio and high serum creatinine (SCr); the independent predictors of larger kidney volume (>97.5 percentile for age and sex) were younger age, female sex, diabetes, obesity, high height, high waist:hip ratio and lower SCr. Estimated heritability for kidney volume was 15%, and for length 27%; kidney volume correlated strongly with birthweight. CONCLUSIONS: Overall, in a general healthy population, kidney measures declined with age differently in men and women. The determinants of kidney parenchymal volume include genetic factors and modifiable clinical factors.

Arterial stiffness and multiple organ damage: a longitudinal study in population.

AIMS: Previous cross-sectional observation identified arterial aging, indexed as pulse-wave velocity (PWV), as a key determinant of the simultaneous multiple organ damage (heart, carotid artery, and kidney). The aim of the present cohort study is to investigate trajectories of repeated measures of PWV and traditional CV risk factors in subjects who eventually presented clinical evidence of multiple organ damage in the SardiNIA study. METHODS AND RESULTS: Organ damage was measured in the heart (left ventricular hypertrophy, LVH), the common carotid artery (intima-media thickness > 0.9 mm and/or plaque), and the kidney (eGFR < 60 ml/min/1.73 m2) of 2130 men and women of a broad age range participating the SardiNIA study. SHATS was defined as the simultaneous occurrence of all the three-organ damages. Trajectory in traditional CV risk factors and PWV was analyzed retrospectively (four observations over 9 years) according to the number of organ damage (from 0 to 3). Compared to subjects with no organ damage, after controlling for traditional CV risk factors, each 1 m/s increase in baseline PWV was accompanied by a 93% higher odds of developing SHATS; and each 1 cm/s (0.01 m/s) annual increase in PWV by a 31% greater odds of developing SHATS. CONCLUSIONS: Arterial stiffness, a proxy of arterial aging that can be measured clinically as PWV, is an integrated predictive marker of multiple age-associated organ damage recognized as clinical diseases.

CD8+CD28-CD127loCD39+ regulatory T-cell expansion: A new possible pathogenic mechanism for HIV infection?

BACKGROUND: HIV-associated immunodeficiency is related to loss of CD4+ T cells. This mechanism does not explain certain manifestations of HIV disease, such as immunodeficiency events in patients with greater than 500 CD4+ T cells/µL. CD8+CD28-CD127loCD39+ T cells are regulatory T (Treg) lymphocytes that are highly concentrated within the tumor microenvironment and never analyzed in the circulation of HIV-infected patients. OBJECTIVES: We sought to analyze the frequency of CD8+CD28-CD127loCD39+ Treg cells in the circulation of HIV-infected patients. METHODS: The frequency of circulating CD8+CD28-CD127loCD39+ Treg cells was analyzed and correlated with viral load and CD4+ T-cell counts/percentages in 93 HIV-1-infected patients subdivided as follows: naive (n = 63), elite controllers (n = 19), long-term nonprogressors (n = 7), and HIV-infected patients affected by tumor (n = 4). The same analyses were performed in HIV-negative patients with cancer (n = 53), hepatitis C virus-infected patients (n = 17), and healthy donors (n = 173). RESULTS: HIV-infected patients had increased circulating levels of functional CD8+CD28-CD127loCD39+ Treg cells. These cells showed antigen specificity against HIV proteins. Their frequency after antiretroviral therapy (ART) correlated with HIV viremia, CD4+ T-cell counts, and immune activation markers, suggesting their pathogenic involvement in AIDS- or non-AIDS-related complications. Their increase after initiation of ART heralded a lack of virologic or clinical response, and hence their monitoring is clinically relevant. CONCLUSION: HIV infection induces remarkable expansion of CD8+CD28-CD127loCD39+ Treg cells, the frequency of which correlates with both clinical disease and signs of chronic immune cell activation. Monitoring their frequency in the circulation is a new marker of response to ART when effects on viremia and clinical response are not met.

A meta-analysis of 120 246 individuals identifies 18 new loci for fibrinogen concentration.

Genome-wide association studies have previously identified 23 genetic loci associated with circulating fibrinogen concentration. These studies used HapMap imputation and did not examine the X-chromosome. 1000 Genomes imputation provides better coverage of uncommon variants, and includes indels. We conducted a genome-wide association analysis of 34 studies imputed to the 1000 Genomes Project reference panel and including ∼120 000 participants of European ancestry (95 806 participants with data on the X-chromosome). Approximately 10.7 million single-nucleotide polymorphisms and 1.2 million indels were examined. We identified 41 genome-wide significant fibrinogen loci; of which, 18 were newly identified. There were no genome-wide significant signals on the X-chromosome. The lead variants of five significant loci were indels. We further identified six additional independent signals, including three rare variants, at two previously characterized loci: FGB and IRF1. Together the 41 loci explain 3% of the variance in plasma fibrinogen concentration.

Adipocytokine correlations with thyroid function and autoimmunity in euthyroid sardinians.

OBJECTIVE: Cytokines release by adipocytes could interact with TSH secretion. We evaluated the relationship between adipocytokines and TSH. We further tested for association of cytokines and thyroid autoimmunity. METHODS: We conducted a cross-sectional study in a community-based sample including 5385 individuals (2964 female) with TSH within the reference range. Subjects who reported taking thyroid medications or drugs that alter thyroid function were excluded. TSH, FT4, adiponectin, leptin, antibody against thyroperoxidase and against thyroglobulin were measured. Linear and logistic regression models were used to test for association. RESULTS: Females had higher adiponectin and leptin level and increased frequency of thyroid antibodies. In multiple regression analysis TSH was directly associated with leptin (ß = 0.003, p = 0.001) and the presence of circulating antibody against thyroperoxidase (ß = 0.315, p < 0.001), but negatively associated with age (ß = -0.012, p < 0.001) and FT4 (ß = -0.359, p < 0.001). Adiponectin, the presence of antibody against thyroglobulin and smoking habit were not associated with TSH levels (p = 0.223, p = 0.174 and p = 0.788, respectively). Logistic regression analysis revealed that higher adiponectin levels were associated with thyroid autoimmunity. CONCLUSIONS: Leptin is positively associated with TSH levels in euthyroid individuals, suggesting an effect of the adipokine on TSH secretion. Our results support the hypothesis that the leptin and pituitary-thyroid axis might interact in the context of energy homeostasis. The effect of adiponectin on thyroid autoimmunity will require more studies.

Glucose-6-phosphate dehydrogenase deficiency accelerates arterial aging in diabetes.

AIMS: High glucose levels and Glucose-6-Phosphate Dehydrogenase deficiency (G6PDd) have both tissue inflammatory effects. Here we determined whether G6PDd accelerates arterial aging (information linked stiffening) in diabetes. METHODS: Plasma glucose, interleukin 6 (IL6), and arterial stiffness (indexed as carotid-femoral Pulse Wave Velocity, PWV) and red blood cell G6PD activity were assessed in a large (4448) Sardinian population. RESULTS: Although high plasma glucose in diabetics, did not differ by G6DP status (178.2 ± 55.1 vs 169.0 ± 50.1 mg/dl) in G6DPd versus non-G6PDd subjects, respectively, IL6, and PWV (adjusted for age and glucose) were significantly increased in G6PDd vs non-G6PDd subjects (PWV, 8.0 ± 0.4 vs 7.2 ± 0.2 m/sec) and (IL6, 6.9 ± 5.0 vs 4.2 ± 3.0 pg/ml). In non-diabetics, neither fasting plasma glucose, nor IL6, nor PWV were impacted by G6PDd. CONCLUSION: G6PDd in diabetics is associated with increased inflammatory markers and accelerated arterial aging.

Transcriptome organization of white blood cells through gene co-expression network analysis in a large RNA-seq dataset.

Gene co-expression network analysis enables identification of biologically meaningful clusters of co-regulated genes (modules) in an unsupervised manner. We present here the largest study conducted thus far of co-expression networks in white blood cells (WBC) based on RNA-seq data from 624 individuals. We identify 41 modules, 13 of them related to specific immune-related functions and cell types (e.g. neutrophils, B and T cells, NK cells, and plasmacytoid dendritic cells); we highlight biologically relevant lncRNAs for each annotated module of co-expressed genes. We further characterize with unprecedented resolution the modules in T cell sub-types, through the availability of 95 immune phenotypes obtained by flow cytometry in the same individuals. This study provides novel insights into the transcriptional architecture of human leukocytes, showing how network analysis can advance our understanding of coding and non-coding gene interactions in immune system cells.

Echocardiographic heart ageing patterns predict cardiovascular and non-cardiovascular events and reflect biological age: the SardiNIA study.

AIMS: Age is a crucial risk factor for cardiovascular (CV) and non-CV diseases. As people age at different rates, the concept of biological age has been introduced as a personalized measure of functional deterioration. Associations of age with echocardiographic quantitative traits were analysed to assess different heart ageing rates and their ability to predict outcomes and reflect biological age. METHODS AND RESULTS: Associations of age with left ventricular mass, geometry, diastolic function, left atrial volume, and aortic root size were measured in 2614 healthy subjects. Based on the 95% two-sided tolerance intervals of each correlation, three discrete ageing trajectories were identified and categorized as 'slow', 'normal', and 'accelerated' heart ageing patterns. The primary endpoint included fatal and non-fatal CV events, and the secondary endpoint was a composite of CV and non-CV events and all-cause death. The phenotypic age of the heart (HeartPhAge) was estimated as a proxy of biological age. The slow ageing pattern was found in 8.7% of healthy participants, the normal pattern in 76.9%, and the accelerated pattern in 14.4%. Kaplan-Meier curves of the heart ageing patterns diverged significantly (P = 0.0001) for both primary and secondary endpoints, with the event rate being lowest in the slow, intermediate in the normal, and highest in the accelerated pattern. In the Cox proportional hazards model, heart ageing patterns predicted both primary (P = 0.01) and secondary (P = 0.03 to <0.0001) endpoints, independent of chronological age and risk factors. Compared with chronological age, HeartPhAge was 9 years younger in slow, 4 years older in accelerated (both P < 0.0001), and overlapping in normal ageing patterns. CONCLUSION: Standard Doppler echocardiography detects slow, normal, and accelerated heart ageing patterns. They predict CV and non-CV events, reflect biological age, and provide a new tool to calibrate prevention timing and intensity.

Age is the main risk factor for cardiovascular (CV) disease. Since people age and develop diseases at very different rates, biological age has been proposed as a more accurate measure of the body's functional decline. This study aimed to investigate the ageing rates of the heart and to assess their impact on CV events. The phenotypic age of the heart was also estimated as a proxy for biological age. Associations of age with Doppler echocardiographic parameters were analysed in a subgroup of 2614 clinically healthy subjects, part of a larger cohort of 3817 adults of both sexes.Three patterns of slow, normal, and accelerated ageing rates of the heart were detected. They predicted both CV and non-CV events, with different and progressively increasing event rates from the slow to the accelerated pattern. Compared with chronological age, the phenotypic (biological) age of the heart was 9 years younger in the slow pattern, 4 years older in the accelerated pattern, and comparable in the normal pattern.A standard Doppler echocardiogram is therefore able to detect three distinct heart ageing patterns, which reflect different biological susceptibilities to age-dependent diseases and provide a new tool for personalizing timeliness and intensity of prevention.