Circulating immune cells are frequently phenotyped by flow cytometry starting from frozen samples. However, cryopreservation can affect marker expression and cell recovery. To understand which antigens are detectable and reliable after sample cryopreservation, we compared 438 antibodies measured on B, T, and NK-enriched cells and monocytes in frozen lympho-monocytes and blood with the corresponding fresh blood samples. Cryopreservation affected the expression of 283 markers in lympho-monocytes and 262 in blood, modifying them by more than 20% with respect to fresh blood. Thus, it is essential to carefully evaluate antibody performance when working with frozen samples. To maximize the usability of our results, make them publicly accessible and ready to visualize, we created a catalogue of marker expression variability before and after freezing, namely FlowCLOc. This catalogue simplifies flow cytometry panel design, reducing time-consuming preliminary tests to select the most appropriate and specific markers for staining both frozen and fresh samples.

Background: Sarcopenia is the age-related, progressive loss of strength, function, and skeletal muscle mass, which can be assessed with specific tests. The Growth differentiation factor 15 (GDF-15) has been proposed as a key biomarker of aging, and it has been associated with mitochondrial dysfunction, cachexia, and physical impairment. Methods: The cohort of this study comes from the SardiNIA study, an ongoing longitudinal survey focused on the identification of genetic and phenotypic variants associated with aging. We assessed hand grip strength, gait speed, and GDF-15 in all samples. Linear multivariate analysis was used to assess the correlation after adjusting for a range of potential confounders. Results: The sample consisted of 4842 subjects (57.5% female) with a median age of 48.6 years. Levels of GDF-15 were comparable between males and females and showed a strong positive association with aging (rho 0.617, p < 0.001). Linear multivariate regression analyses showed that GDF-15 was negatively associated with gait speed and grip strength in both hands (respectively, Beta -0.09, Beta -0.07, and Beta -0.08, p < 0.001 for all). Conclusions: GDF-15 was negatively associated with physical function. GDF-15 may be considered a proxy for reduced physical performance. Future research is needed to understand the pathogenetic role of GDF-15 in the reduction in skeletal muscle in aging people.

Clonal haematopoiesis (CH) arises from the expansion of hematopoietic stem cells (HSCs) carrying leukaemia-associated somatic mutations. CH is linked to pathological immune dysregulation and a greater risk of age-related inflammatory diseases. Yet, how CH mutations impact HSC differentiation into immune effector cells remains understudied. Here, we report a single-cell resolution functional and multi-omic investigation of HSC clonal and differentiation dynamics in individuals with DNMT3A-R882 CH. DNMT3A- R882 reshapes the clonal architecture of haematopoiesis towards an aged phylogenetic structure. Functionally, DNMT3A- R882 HSCs produce decreased monocytic output but more abundant and mature neutrophil progeny compared to WT HSCs in the same individual. Whereas DNMT3A- R882 myeloid progenitors display attenuated inflammatory transcriptional programmes, DNMT3A- R882 mature neutrophils acquire proinflammatory and immunomodulatory features typical of maladaptive immunity and CH co-morbidities. Our findings, validated in humanised mice, identify aberrant DNMT3A -R882 HSC-driven neutropoiesis as a key link between CH, immune dysregulation and risk of inflammatory disease. ### Competing Interest Statement EL receives grant funding from CSL Behring for research independent to that presented here. MAF is a current employee and stockholder of AstraZeneca. GSV is a consultant to STRM.BIO and holds a research grant from AstraZeneca for research unrelated to that presented here. LS is a shareholder of Sequentify and Cliseq. British Council BIRAX, 47BX16ELLS Wellcome Trust, https://ror.org/029chgv08, 107630/Z/15/Z, 203151/Z/16/Z, CC20-27 Blood Cancer United, 7035-24 Blood Cancer UK, https://ror.org/0055acf80, 24008 Medical Research Council, 203151/Z/16/Z, CC2027 Alborada Trust, https://ror.org/00c9bhj04 Israel Science Foundation, 3165/19, 2427/18, 1123/21 Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine European Research Council, UDEMC, 633964 The WBH foundation Gates Cambridge Trust, 10350885 Cancer Research UK, CTRQQR-2021\100012, CC2027 Kay Kendall Leukaemia Fund, KKL 1397 European Hematology Association NIH Common Fund, https://ror.org/001d55x84, N01-AG-1-2109, HHSN271201100005C

BackgroundThe immunoglobulin level following vaccination against SARS-CoV-2 results from a multifaceted immunological process involving cells and molecules that determine its efficacy and protect us against severe infection outcomes. The observed heterogeneity in the immune response to vaccination is partly attributable to host genetic factors; however, this genetic contribution has only been partially explored.MethodsTo elucidate the mechanisms underlying the antibody response elicited by the Pfizer-BioNTech vaccine, we conducted a genome-wide association study on anti-spike immunoglobulin G levels measured in 1,968 Italian individuals who received two doses of the vaccine, selected from a larger cohort of 7,169 volunteers characterized for 8 million genetic variants. Sex, age, body mass index, smoking habit, and time elapsed between vaccine administration and blood draw were accounted as covariates in the linear regression model.ResultsWe identified a novel signal of association on chromosome 3 in the intronic region of the CMTM8 gene and confirmed one previously identified at the HLA locus close to the HLA-B gene. The lead SNP in the CMTM8 gene, rs7643677 (p-value = 2.095×10-8), is associated with anti-S IgG levels and with the expression level of CD66b on granulocytic/polymorphonuclear myeloid-derived suppressor cells.ConclusionsThese findings support a role for CMTM8 in regulating the suppressive activity of specific immune cells, and suggest a potential interplay among genetic, humoral, and cellular mechanisms underlying the immune response to SARS-CoV-2 vaccination.

Personality traits describe stable differences in how people think, feel and behave, and how they interact with and experience their social and physical environments1,2. Many questions remain unanswered about associations between DNA and personality traits, such as their robustness, their generalizability and the biological and social pathways through which they act. Here we meta-analyse data across 46 cohorts comprising 611,037 to 1.14 million participants with European-like and African-like genomes for genome-wide association studies (GWAS) of the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism and openness to experience), and data from up to 50,725 participants for within-family GWAS. We identify 1,260 lead genetic variants associated with personality, including 824 novel variants3. Common genetic variants explain a moderate 4.8-9.3% of the variance in measures of each trait, and 9.3-13.3% among instruments with typical measurement reliability. Genetic associations with personality are highly consistent but not identical across geography, reporter (self versus close other), age group and measurement instrument, and we find minimal spousal assortment for personality in recent history. In contrast to many other social and behavioural traits4,5, within-family GWAS and polygenic index analyses indicate that genetic associations with personality are minimally confounded by the shared family environment. Polygenic prediction, genetic correlation and Mendelian randomization analyses indicate that personality traits have widespread, potentially causal associations with consequential behaviours and life outcomes. Overall, we find that the genetic architecture of personality is robustly generalizable, minimally confounded and widely relevant to human experience.

BACKGROUND:The relation between thyroid hormone and bone microarchitecture at tibial level is not clear. This study aimed to assessthe correlation between thyrotropin and free thyroxine in a large cohort of participants from the SardiNIA study. METHODS:Peripheral quantitative computed tomography (pQCT) was assessed on 4413 participants (55.0% female). Parameters of pQCT were analyzed separately as exposure variables, while thyroid hormones (thyrotropin and free thyroxine) were treated as independent variables in multivariate regression models. RESULTS:After adjusting for confounders, free thyroxine was negatively associated with both total bone density at 4% level (coefficient - 7.26, standard error 2.78, p = 0.009) and cortical bone density at 38% level (coefficient - 11.44, standard error 3.57, p = 0.001). FT4 had also a positive association with eccentricity index at 38% level (coefficient 0.017, standard error 0.01, p = 0.008). TSH showed a positive association with total and trabecular bone density at 4% level (respectively, coefficient 0.79, standard error 0.34, p = 0.021 and coefficient 0.83, standard error 0.31, p = 0.008). CONCLUSION:Thyroid hormones are associated with a weaker bone geometry at tibia level. This finding may support the hypothesis that excess of thyroid hormones has deleterious effect on bone microarchitecture. Further studies are needed to determine whether targeted treatment could help mitigate bone loss in patients with thyroid disorders.

Background An association between glucose‐6‐phosphate dehydrogenase deficiency (G6PDdef) and arterial stiffness has cross sectionally been reported in diabetic, but not in nondiabetic, subjects in Sardinia. The aims of the present longitudinal study were to evaluate: (1) sex differences in the influence of G6PDdef on trajectory of arterial stiffness; (2) the impact of hyperglycemia on trajectory of arterial stiffness; and (3) mechanistic pathways underlying this influence. Methods In the Sardinia Study, repeated measurements of pulse‐wave velocity were analyzed to characterize trajectories of arterial stiffness in >4000 men and women, aged 20 to 100 years, during a 9.4‐year follow‐up. Results In men with high glucose, G6PDdef was associated with a steeper longitudinal increase in pulse‐wave velocity (yearly change: 12.5 versus 6.9 cm/s in those without G6PDdef). In contrast, in women with high glucose, the longitudinal change is not influenced by the presence or absence of G6PDdef (yearly change: 4.5 cm/s in both groups). With high glucose, men with G6PDdef showed similar trends in fat deposition, but no substantial increase in triglycerides, over time compared with those without G6PDdef. Conversely, women with G6PDdef showed a greater gluteofemoral fat deposition over time, with no increase in visceral fat deposition, and a similar trajectory in triglycerides compared with those without G6PDdef. Conclusions Lower G6PD activity is associated with an accelerated increase in arterial stiffness in men, but not in women. This effect is accompanied by sex‐specific changes in metabolic factors. Future studies will characterize the link between metabolic reprogramming and changes in arterial stiffness.

The severity of malaria varies substantially between individuals, but the mechanisms that underlie these differences remain unclear. Because erythrocytes have a key role in malaria biology, genetic variants associated with the development of these cells could inform the mechanisms that determine disease severity. Here we investigate the mechanistic basis of the association of the variant rs112233623-T with erythrocyte properties, and examine its role in modulating malaria severity. This variant is associated with increased levels of haemoglobin A2, increased erythrocyte size and reduced erythrocyte number1,2. It is found in an erythroid enhancer of CCND3, which encodes cyclin D3-a cell-division activator that enhances the pentose phosphate pathway and thereby helps to counteract reactive oxygen species (ROS)3. We show that rs112233623-T disrupts a binding site for the transcription factor SMAD3, weakens enhancer activity and, in erythrocyte precursors (erythroblasts), is associated with reduced CCND3 expression and inhibition of the G1-S cell-cycle transition, concomitant with a reduction in the number of erythrocytes and an increase in their size. Using population genetic methods, we observe signatures of positive selection for rs112233623-T in the genetic history of Sardinia, a region in which malaria was once prevalent. Furthermore, we show that parasite growth is impaired in cultured Plasmodium falciparum-infected erythrocytes from rs112233623-T carriers, and that this impairment correlates with ROS levels. This mirrors our observations in erythrocytes from individuals who are deficient in the pentose-phosphate-pathway enzyme G6PD-a trait associated with protection against malaria in some settings-and highlights a common ROS-based mechanism of malaria resistance. Our results suggest that a reduction in CCND3 in erythroblasts constitutes a mechanism of resistance to malaria, and could enable therapeutic interventions.

The TYK2:p.Pro1104Ala (rs34536443) hypomorph variant has been associated with protection against numerous autoimmune disorders. Thus, its mechanism of action becomes of great interest. Here, consistent with the participation of activated immune cells in autoimmunity, we show that the variant regulates the levels of immune cells at a human, general population level and is associated particularly with higher levels of T and B lymphocytes, especially the naïve (non-activated) compartment. Also, consistent with a protective function in autoimmunity, the level of regulatory CD4+ T cells was increased. Thus, this variant decreases immune activation thereby protecting from autoimmunity. Our work links the cellular mechanism regulated by the TYK2:p.Pro1104Ala variant to autoimmunity protection and supports TYK2 as a therapeutic target in autoimmunity.

Genotype-phenotype associations can be context-dependent and dynamic in nature leading to heterogeneity of genetic effects across different parts of the phenotype distribution. Quantile regression, an alternative to linear regression for continuous phenotypes, is particularly well suited for detecting and characterizing heterogeneous genotype-phenotype associations. Here we propose a novel and computationally efficient whole-genome quantile regression technique, Regenie.QRS, for biobank-scale GWAS data with genetic structure. Our approach first estimates the polygenic effect, and then incorporates this effect as an offset in the non-mixed quantile regression model. Our simulations demonstrate robust control of type I error and higher power to detect heterogeneous associations relative to linear regression in GWAS, and improved power over the marginal quantile regression tests. We present applications using data from the UK Biobank and the ProgeNIA/SardiNIA project, where we show the advantages of Regenie.QRS in identifying and characterizing heterogeneous genetic effects. To cite just one interesting example, using quantile regression we are able to show that even though variants at the G6PC2 locus increase glucose levels, their effects are much stronger at lower quantiles of glucose level distribution than at higher quantiles, showing that G6PC2 serves as a guardian against low glucose levels without driving dangerous hyperglycemia, which may explain the lack of association with diabetes risk.

Clonal hematopoiesis (CH) is characterized by expanding blood cell clones carrying somatic mutations in healthy aged individuals and is associated with various age-related diseases and all-cause mortality. While CH mutations affect diverse genes associated with myeloid malignancies, their mechanisms of expansion and disease associations remain poorly understood. We investigate the relationship between clonal fitness and clinical outcomes by integrating data from three longitudinal aging cohorts (n=713, observations=2,341). We demonstrate pathway-specific fitness advantage and clonal composition influence clonal dynamics. Further, the timing of mutation acquisition is necessary to determine the extent of clonal expansion reached during the host individual’s lifetime. We introduce MACS120, a metric combining mutation context, timing, and variant fitness to predict future clonal growth, outperforming traditional variant allele frequency measurements in predicting clinical outcomes. Our unified analytical framework enables standardized clonal dynamics inference across cohorts, advancing our ability to predict and potentially intervene in CH-related pathologies.

Background: Blood pressure is associated with overt thyroid disorders, but the role of subclinical diseases is not clear, particularly when blood pressure is assessed at the clinical office. Ambulatory blood pressure monitoring over 24 h provides additional clinical information, which correlates with many cardiovascular endpoints. The aim of our work is to examine whether thyroid function is related to systolic and diastolic blood pressure assessed by ambulatory blood pressure monitoring. Methods: We enrolled 3277 subjects from the SardiNIA project. Thyroid function and ambulatory blood pressure monitoring were assessed in all the participants. Results: TSH was associated with average 24 h and daytime DBP in males but not in females, after adjusting for confounders (respectively, Coef -0.192 p = 0.025, and Coef. -0.021, p = 0.018). We found no association between TSH and DBP or SBP during nighttime. Conclusions: Low TSH in males is positively associated with high DBP. Further studies of underlying mechanisms will need to explore our findings.

Personality traits describe stable differences in how individuals think, feel, and behave and how they interact with and experience their social and physical environments. We assemble data from 46 cohorts including 611K-1.14M participants with European-like and African-like genomes for genome-wide association studies (GWAS) of the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), and data from 51K participants for within-family GWAS. We identify 1,257 lead genetic variants associated with personality, including 823 novel variants. Common genetic variants explain 4.8%-9.3% of the variance in each trait, and 10.5%-16.2% accounting for measurement unreliability. Genetic effects on personality are highly consistent across geography, reporter (self vs. close other), age group, and measurement instrument, and we find minimal spousal assortment for personality in recent history. In stark contrast to many other social and behavioral traits, within-family GWAS and polygenic index analyses indicate little to no shared environmental confounding in genetic associations with personality. Polygenic prediction, genetic correlation, and Mendelian randomization analyses indicate that personality genetics have widespread, potentially causal associations with a wide range of consequential behaviors and life outcomes. The genetic architecture of personality is robust and fundamental to being a human.

Subtle population structure remains a significant concern in genome-wide association studies. Using human height as an example, we show how quantile regression, a natural extension of linear regression, can better correct for subtle population structure due to its inherent ability to adjust for quantile-specific effects of covariates such as principal components. We utilize data from the UK biobank and the SardiNIA/ProgeNIA project for demonstration.

Polygenic risk scores (PRS) are widely used in post-GWAS analyses to predict complex traits across humans, animals, and plants. While significant progress has been made in developing new PRS methods, much less attention has been given to quantifying the uncertainty associated with these predictions. In this work, we propose a method for individualized uncertainty quantification based on quantile regression. When paired with conformal prediction, this approach enables the construction of prediction intervals with guaranteed coverage, offering lower and upper bounds within which the phenotype is likely to fall with high probability. We apply this framework to data from the UK Biobank and the ProgeNIA/SardiNIA studies, showing that the resulting prediction intervals: (1) maintain valid coverage under minimal model assumptions, (2) provide more realistic individualized estimates of uncertainty by allowing for asymmetry and individual-specific interval lengths, and (3) exhibit reduced uncertainty compared to existing methods. Overall, we present a novel framework for individualized uncertainty quantification in PRS analyses and highlight the importance of incorporating uncertainty into predictive modeling. ### Competing Interest Statement The authors have declared no competing interest.

The mechanisms through which mutations in splicing factor genes drive clonal hematopoiesis (CH) and myeloid malignancies, and their close association with advanced age, remain poorly understood. Here we show that telomere maintenance plays an important role in this phenomenon. First, by studying 454,098 UK Biobank participants, we find that, unlike most CH subtypes, splicing-factor-mutant CH is more common in those with shorter genetically predicted telomeres, as is CH with mutations in PPM1D and the TERT gene promoter. We go on to show that telomere attrition becomes an instrument for clonal selection in advanced age, with splicing factor mutations 'rescuing' HSCs from critical telomere shortening. Our findings expose the lifelong influence of telomere maintenance on hematopoiesis and identify a potential shared mechanism through which different splicing factor mutations drive leukemogenesis. Understanding the mechanistic basis of these observations can open new therapeutic avenues against splicing-factor-mutant CH and hematological or other cancers.

Growth differentiation factor 15 (GDF15) is a stress-response cytokine, which exerts different actions in physiological and pathological conditions. Thyroid disorders are common in the general population and their role on GDF15 levels has not been sufficiently addressed. Serum levels of GDF-15 and thyroid function were assessed in a large sample from the SardiNIA cohort (n = 4413). We further collected antibodies against thyroperoxidase and anti-thyroglobulin in all participants. Thyroid function correlated with GDF15. Specifically, after adjusting for covariates, thyrotropin had a positive association with GDF15, while free thyroxine was negatively correlated. We also found that subjects with circulating antibodies against thyroperoxidase had lower GDF15 levels. The reduced level of the thyroid hormone can cause a decreased metabolic activity and higher cell stress response, which, in turn, increases the production of GDF-15 as a defense mechanism. The association between antibodies against thyroperoxidase and GDF15 deserves additional study to elucidate the pathophysiologic relationship.