Case-control study of adverse childhood experiences and multiple sclerosis risk and clinical outcomes

Background Adverse childhood experiences (ACEs) are linked to numerous health conditions but understudied in multiple sclerosis (MS). This study’s objective was to test for the association between ACEs and MS risk and several clinical outcomes. Methods We used a sample of adult, non-Hispanic MS cases (n = 1422) and controls (n = 1185) from Northern California. Eighteen ACEs were assessed including parent divorce, parent death, and abuse. Outcomes included MS risk, age of MS onset, Multiple Sclerosis Severity Scale score, and use of a walking aid. Logistic and linear regression estimated odds ratios (ORs) (and beta coefficients) and 95% confidence intervals (CIs) for ACEs operationalized as any/none, counts, individual events, and latent factors/patterns. Results Overall, more MS cases experienced ≥1 ACE compared to controls (54.5% and 53.8%, respectively). After adjusting for sex, birthyear, and race, this small difference was attenuated (OR = 1.01, 95% CI: 0.87, 1.18). There were no trends of increasing or decreasing odds of MS across ACE count categories. Consistent associations between individual ACEs between ages 0–10 and 11–20 years and MS risk were not detected. Factor analysis identified five latent ACE factors, but their associations with MS risk were approximately null. Age of MS onset and other clinical outcomes were not associated with ACEs after multiple testing correction. Conclusion Despite rich data and multiple approaches to operationalizing ACEs, no consistent and statistically significant effects were observed between ACEs with MS. This highlights the challenges of studying sensitive, retrospective events among adults that occurred decades before data collection.

Maternal first trimester metabolic profile in TGA pregnancies - a case-control study

Objective: To evaluate the association between maternal first trimester metabolic profile and transposition of the great arteries (TGA) in the offspring. Design: A matched case-control study using national register data and maternal first trimester blood samples. Settings: Finland Patients: A total of 100 mothers with TGA in their offspring and 200 controls matched by the year of birth and sex of the child, and the age and the body mass index (BMI) of the mother. Interventions: None. Main outcome measures: To identify the impact of 73 metabolic measure concentrations on the likelihood that a child would be born with TGA. Results: A higher concentration of four subtypes in extremely large very-low-density lipoprotein (VLDL) particles and one in large VLDL particles were observed in mothers with TGA in their offspring. This finding did not reach statistical significance after multiple testing correction. In the analysis of the subgroups of maternal BMI over 25 and 30 only, the odds ratios (OR) of the metabolic variables were higher compared to the original population. In the group with a BMI over 25 the mean OR was 1.3 (N= 111, total) and in the group with a BMI over 30 was 2.1 (N= 42, total). Conclusions: We found no statistically significant difference between the metabolic profiles of mothers with TGA in their offspring, and their controls. However, the trend in our BMI-matched study potentially indicates that the likelihood of TGA development associates with maternal early-pregnancy metabolic profile, and the association is more pronounced in the subgroups of BMI over 25 and 30. Further studies are needed especially in the higher BMI groups.

Capturing SNP Association across the NK Receptor and HLA Gene Regions in Multiple Sclerosis by Targeted Penalised Regression Models

Conventional genome-wide association studies (GWASs) of complex traits, such as Multiple Sclerosis (MS), are reliant on per-SNP p-values and are therefore heavily burdened by multiple testing correction. Thus, in order to detect more subtle alterations, ever increasing sample sizes are required, while ignoring potentially valuable information that is readily available in existing datasets. To overcome this, we used penalised regression incorporating elastic net with a stability selection method by iterative subsampling to detect the potential interaction of loci with MS risk. Through re-analysis of the ANZgene dataset (1617 cases and 1988 controls) and an IMSGC dataset as a replication cohort (1313 cases and 1458 controls), we identified new association signals for MS predisposition, including SNPs above and below conventional significance thresholds while targeting two natural killer receptor loci and the well-established HLA loci. For example, rs2844482 (98.1% iterations), otherwise ignored by conventional statistics (p = 0.673) in the same dataset, was independently strongly associated with MS in another GWAS that required more than 40 times the number of cases (~45 K). Further comparison of our hits to those present in a large-scale meta-analysis, confirmed that the majority of SNPs identified by the elastic net model reached conventional statistical GWAS thresholds (p < 5 × 10−8) in this much larger dataset. Moreover, we found that gene variants involved in oxidative stress, in addition to innate immunity, were associated with MS. Overall, this study highlights the benefit of using more advanced statistical methods to (re-)analyse subtle genetic variation among loci that have a biological basis for their contribution to disease risk.

networkGWAS: A network-based approach for genome-wide association studies in structured populations

While the search for associations between genetic markers and complex traits has discovered tens of thousands of trait-related genetic variants, the vast majority of these only explain a tiny fraction of observed phenotypic variation. One possible strategy to detect stronger associations is to aggregate the effects of several genetic markers and to test entire genes, pathways or (sub)networks of genes for association to a phenotype. The latter, network-based genome-wide association studies, in particular suffers from a huge search space and an inherent multiple testing problem. As a consequence, current approaches are either based on greedy feature selection, thereby risking that they miss relevant associations, and/or neglect doing a multiple testing correction, which can lead to an abundance of false positive findings. To address the shortcomings of current approaches of network-based genome-wide association studies, we propose <tt>networkGWAS</tt>, a computationally efficient and statistically sound approach to gene-based genome-wide association studies based on mixed models and neighborhood aggregation. It allows for population structure correction and for well-calibrated p-values, which we obtain through a block permutation scheme. <tt>networkGWAS</tt> successfully detects known or plausible associations on simulated rare variants from H. sapiens data as well as semi-simulated and real data with common variants from A. thaliana and enables the systematic combination of gene-based genome-wide association studies with biological network information.

Impact of Tet2 Deficiency, and of TET2 Mutations in Clonal Hematopoiesis, on Neutrophil/Granulocyte Immune Function

BACKGROUND: Neutrophils, the most abundant leukocytes and granulocytes, are important regulators of cardiovascular, inflammatory and infectious diseases, yet their role in the pathophysiology of clonal hematopoiesis of indeterminate potential (CHIP) has not been adequately addressed. The effects of inactivating CHIP-driver mutations in the epigenetic regulator TET2 in neutrophils especially, are broadly unknown. HYPOTHESIS: Tet2 inactivation in murine neutrophils, and TET2 mutations in CHIP in humans (CHIP TET2), perturb granulocyte immune effector functions. METHODS: Neutrophils were obtained (EasySep™, StemCell) from the bone marrow of 2- to 4-months-old, sex-matched, control Tet2 f/f;Vav1-icre - (Tet2 f/f) and hematopoietic knockout Tet2 f/f;Vav1-icre + (Tet2 -/-) mice. Neutrophils were cultured (RPMI+10% mouse serum/FBS) and: i) stained with Mitotracker Deep Red/Nuc Blue, co-cultured and imaged (Leica SP8-X) for 30min with GFP-labeled Staphylococcus aureus (10:1 ratio) and analyzed in FIJI; ii) cultured for 3h with vehicle or 10μg/mL of S. aureus lipotechoic acid (LTA). RNA-Seq was generated (Illumina QuantSeq 3' mRNA, single-end 75bp read lengths, 5 million reads/sample), trimmed, aligned to GRCm39 using STAR. CHIP participant DNA and RNA were sequenced previously from whole blood (Cook et al., Bld Adv 2019; Cook et al., ASH 2018, with a 48-gene panel on Ion Proton, and ribo-depleted bulk RNA on Illumina, respectively). New CHIP TET2 vs. no CHIP, and murine RNA-Seq analyses were carried out in DESeq2. Human serum granule protein levels were quantified by ELISA (VersaMax). Mann-Whitney U tests were carried out in Prism. P&lt;0.05 was considered statistically significant, and Benjamini-Hochberg multiple testing correction was applied as needed. RESULTS: Tet2 -/- mice had 1.34-fold more bone marrow CD11b +Ly6G + neutrophils than control Tet2 f/f mice (p=0.03), consistent with myeloid expansion. Compared to Tet2 f/f, Tet2 -/- neutrophils phagocytosed fewer S. aureus (Fig1A) and moved more slowly (Fig1B). Preliminary data suggest that Tet2 -/- neutrophil extracellular trap (NET) formation in response to S. aureus was also impaired, showing fewer and less extensive NETs (Fig1C). LTA-stimulated gene expression profiles were similar between Tet2 -/- and Tet2 f/f, suggesting pre-existing differences at baseline. Unexpectedly, the most significant GO term enrichment related to upregulated viral response pathways, including interferon-stimulated genes, (e.g. Ifitm1). The cause is unknown, but this is reminiscent of the constitutive interferon response seen in myelodysplastic syndrome (MDS) patients and TET2-mutant hematopoietic stem cells, where epigenetic dysregulation of endogenous retrotransposable elements leads to a viral mimicry response. Tet2 -/- neutrophils also overexpressed Asprv1, a regulator of inflammation ostensibly acquired from a retrotransposon. Interestingly, Ccdc80, which has been linked to Tet2 and Jak2 functions, was most significantly downregulated in Tet2 -/-, along with the Pnpla1 lipid phosphatase. Finally, Tesc, a promoter of granulocytic differentiation, was upregulated in Tet2 -/-, and there were perturbations of genes encoding neutrophil granule contents. Similarly, human RNA-Seq revealed that several leukocyte (de)granulation-related genes (e.g. lactoferrin LTF, myeloperoxidase MPO) were upregulated in CHIP TET2 subjects to those without CHIP, and these corresponded with higher LTF and MPO serum titers in an expanded cohort (Fig1D,E). Finally, there were striking decreases of gene expression associated with cytotoxic (T/NK) human lymphocytes (i.e. GZMM, TRGV8, etc.). Neutrophil, lymphocyte and monocyte counts were not significantly different between the groups. CONCLUSIONS: Tet2-deficient murine neutrophils have compromised immune function, possibly due to differences in pre-stimulus state. TET2-mutation carrying neutrophils in CHIP may exhibit similar abnormalities, as has been previously noted in neutrophils isolated from MDS patients. Indeed, CHIP is now known to associate with increased risk of bacterial and viral infections, and infection risk has also previously been noted for MDS. People with CHIP have elevated peripheral blood serum MPO and LTF levels, suggesting a difference in leukocyte granule biology, likely related to neutrophils. These data aid in understanding how CHIP alters immunity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

240 Discovery of biomarkers of resistance to immune checkpoint blockade in non-small-cell lung cancer (NSCLC) using high-plex digital spatial profiling

BackgroundDespite the clinical effectiveness of Immune Checkpoint Inhibitors (ICI) in lung cancer, only around 20% remain disease free at 5 years. Predictive biomarkers for ICIs are neither sensitive nor specific. Here, we used the GeoMx Digital Spatial Profiler (DSP) (NanoString, Inc.) to analyze high-plex protein in a quantitative and spatially resolved manner from single formalin-fixed paraffin embedded tissue sections toward the goal of identification of new biomarkers with better predictive value.MethodsPre-treatment samples from 56 patients with NSCLC treated with ICI were collected, represented in Yale tissue microarray 471 (YTMA471), and analyzed. A panel of 71 photocleavable oligonucleotide-labeled primary antibodies (NanoString Human IO panel) was used for protein detection. Protein expression was measured in 4 molecularly defined tissue compartments, defined by fluorescence co-localization (tumor [panCK+], leukocytes [CD45+/CD68-], macrophages [CD68+] and an aggregate stromal immune cell compartment, defined as the sum of leukocyte and macrophage expression [panCK-/CD45+/CD68+]) generating 284 variables representing potential predictive biomarkers. Promising candidates were orthogonally validated with Quantitative Immunofluorescence (QIF). Pre-treatment samples from 40 patients with NSCLC (YTMA404) that received ICI, and 174 non-ICI treated operable NSCLC patients (YTMA423) were analyzed to provide independent cohort validation. All statistical testing was performed using a two-sided significance level of α=0.05 and multiple testing correction (Benjamini-Hochberg method, FDR < 0.1).ResultsInitial biomarker discovery on 284 protein variables were generated by univariate analysis using continuous log-scaled data. High PD-L1 expression in tumor cells predicted longer survival (PFS; HR 0.67, p=0.017) and validated the training cohort. We found 4 markers associated with PFS, and 3 with OS in the stromal compartment. Of these, expression of CD66b in stromal immune cells predicted significantly shorter OS (HR 1.31, p=0.016) and shorter PFS (HR 1.24, p = 0.04). Tertile analysis using QIF on all three tissue cohorts for CD66b expression, assessed by QIF, showed that CD66b was indicative but not prognostic for survival [discovery cohort, YTMA471 (OS; HR 3.02, p=0.013, PFS; HR 2.38, p=0.023), validation cohort; YTMA404 (OS; HR 2.97, p=0.018, PFS; HR 1.85, p=0.1), non-ICI treated cohort YTMA423 (OS; HR 1.02, p>0.9, PFS; HR 0.72, p=0.4)].ConclusionsUsing the DSP technique, we have discovered that CD66b expressed in the stromal immune [panCK-/CD45+/CD68+] molecular compartment is associated with resistance to ICI therapy in NSCLC. This observation was validated by an orthogonal approach in an independent ICI treated NSCLC cohort. Since CD66b identifies neutrophils, further studies are warranted to characterize the role of neutrophils in ICI resistance.AcknowledgementsDr Moutafi is supported by a scholarship from the Hellenic Society of Medical Oncologists (HESMO)Ethics ApprovalAll tissue samples were collected and used under the approval from the Yale Human Investigation Committee protocol #9505008219 with an assurance filed with and approved by the U.S. Department of Health and Human Services

Validation of GWAS-Identified Variants for Anti-TNF Drug Response in Rheumatoid Arthritis: A Meta-Analysis of Two Large Cohorts

We aimed to validate the association of 28 GWAS-identified genetic variants for response to TNF inhibitors (TNFi) in a discovery cohort of 1361 rheumatoid arthritis (RA) patients monitored in routine care and ascertained through the REPAIR consortium and DANBIO registry. We genotyped selected markers and evaluated their association with response to TNFi after 6 months of treatment according to the change in disease activity score 28 (ΔDAS28). Next, we confirmed the most interesting results through meta-analysis of our data with those from the DREAM cohort that included 706 RA patients treated with TNFi. The meta-analysis of the discovery cohort and DREAM registry including 2067 RA patients revealed an overall association of the LINC02549rs7767069 SNP with a lower improvement in DAS28 that remained significant after correction for multiple testing (per-allele ORMeta=0.83, PMeta=0.000077; PHet=0.61). In addition, we found that each copy of the LRRC55rs717117G allele was significantly associated with lower improvement in DAS28 in rheumatoid factor (RF)-positive patients (per-allele ORMeta=0.67, P=0.00058; PHet=0.06) whereas an opposite but not significant effect was detected in RF-negative subjects (per-allele ORMeta=1.38, P=0.10; PHet=0.45; PInteraction=0.00028). Interestingly, although the identified associations did not survive multiple testing correction, the meta-analysis also showed overall and RF-specific associations for the MAFBrs6071980 and CNTN5rs1813443 SNPs with decreased changes in DAS28 (per-allele ORMeta_rs6071980 = 0.85, P=0.0059; PHet=0.63 and ORMeta_rs1813443_RF+=0.81, P=0.0059; PHet=0.69 and ORMeta_rs1813443_RF-=1.00, P=0.99; PHet=0.12; PInteraction=0.032). Mechanistically, we found that subjects carrying the LINC02549rs7767069T allele had significantly increased numbers of CD45RO+CD45RA+ T cells (P=0.000025) whereas carriers of the LINC02549rs7767069T/T genotype showed significantly increased levels of soluble scavengers CD5 and CD6 in serum (P=0.00037 and P=0.00041). In addition, carriers of the LRRC55rs717117G allele showed decreased production of IL6 after stimulation of PBMCs with B burgdorferi and E coli bacteria (P=0.00046 and P=0.00044), which suggested a reduced IL6-mediated anti-inflammatory effect of this marker to worsen the response to TNFi. In conclusion, this study confirmed the influence of the LINC02549 and LRRC55 loci to determine the response to TNFi in RA patients and suggested a weak effect of the MAFB and CNTN5 loci that need to be further investigated.

Shared Genomic Architectures of Covid-19 and Antisocial Behavior: Implications During Pandemics

BACKGROUND. Norm violation, aggression, and antisocial behaviors (ASB) are harmful to society. In times of crisis, such as the current pandemic, individuals with higher antisocial tendencies may subvert efforts to ameliorate social problems. Complicating research on this topic, however, is the fact that variance in both ASB and health traits is partly heritable, suggesting the possibility of genetic correlations between them. METHODS. We characterized the shared polygenic architecture of ASB, Covid-19, and related traits, leveraging summary statistics from genome-wide association studies. RESULTS. After multiple-testing correction, ASB was genetically correlated with average income (rg=-0.54; 95% confidence interval [CI]: -0.65, -0.43); education years (rg=-0.48; CI: -0.59, -0.38; verbal reasoning (rg=-0.44; CI: -0.58, -0.30); healthspan (rg=-0.47; CI: -0.62, -0.31), lifespan (rg=-0.33 (CI: -0.46, -0.21); breastfed as baby (rg=-0.24; 95% CI: -0.38, -0.11); cheese intake (rg=-0.28 (CI: -0.38, -0.18); Covid-19 (rg=0.51, 95% CI: 0.12, 0.90; heavy, manual labor (rg=0.58; CI: 0.45, 0.70); noisy workplace (rg=0.63; CI: 0.48, 0.77); Townsend Deprivation Index (rg=0.70; CI: 0.56, 0.84); gastrointestinal diseases (rg=0.46; 95% CI: 0.23, 0.70); chronic obstructive pulmonary disease (rg=0.51; CI: 0.33, 0.68); genitourinary diseases (rg=0.38; CI: 0.22, 0.55); neuroticism (rg=0.29; CI: 0.20, 0.38); seen doctor for nerves, anxiety, tension, or depression (rg=0.42; CI: 0.31, 0.54); plays computer games (rg=0.15; CI: 0.06, 0.25); violent-crime victim (rg=0.36; CI: 0.16, 0.56); risk tolerance (rg=0.50; CI: 0.39, 0.65); saw sudden violent death (rg=0.42; CI: 0.20, 0.64). CONCLUSIONS. Our results suggest ASB shares genetic architecture with Covid-19 and related health outcomes. We discuss the public-health and bioethical implications of our results.

Polygenic association with severity and long-term outcome in eating disorder cases

About 20% of individuals with anorexia nervosa (AN) remain chronically ill. Therefore, early identification of poor outcome could improve care. Genetic research has identified regions of the genome associated with AN. Patients with anorexia nervosa were identified via the Swedish eating disorder quality registers Stepwise and Riksät and invited to participate in the Anorexia Nervosa Genetics Initiative. First, we associated genetic information longitudinally with eating disorder severity indexed by scores on the Clinical Impairment Assessment (CIA) in 2,843 patients with lifetime AN with or without diagnostic migration to other forms of eating disorders followed for up to 16 years (mean = 5.3 yrs). Second, we indexed development of a severe and enduring eating disorder (SEED) by a high CIA score plus a follow-up time ≥5 years. We associated individual polygenic scores (PGSs) indexing polygenic liability for AN, schizophrenia, and body mass index (BMI) with severity and SEED. After multiple testing correction, only the BMI PGS when calculated with traditional clumping and p value thresholding was robustly associated with disorder severity (βPGS = 1.30; 95% CI: 0.72, 1.88; p = 1.2 x 10-5) across all p value thresholds at which we generated the PGS. However, using the alternative PGS calculation method PRS-CS yielded inconsistent results for all PGS. The positive association stands in contrast to the negative genetic correlation between BMI and AN. Larger discovery GWASs to calculate PGS will increase power, and it is essential to increase sample sizes of the AN GWASs to generate clinically meaningful PGS as adjunct risk prediction variables. Nevertheless, this study provides the first evidence of potential clinical utility of PGSs for eating disorders.

Effects of Cytochrome P450 and Transporter Polymorphisms on the Bioavailability and Safety of Dutasteride and Tamsulosin

Dutasteride and tamsulosin are one of the first-line combination therapies for the management of benign prostatic hyperplasia (BPH). Despite being more effective than monotherapies, they produce frequent adverse drug reactions (ADRs). Institutions such as Food and Drug Administration and European Medicines Agency recommend precaution with CYP2D6 poor metabolizers (PMs) that receive CYP3A4 inhibitors and tamsulosin. However, no specific pharmacogenetic guideline exists for tamsulosin. Furthermore, to date, no pharmacogenetic information is available for dutasteride. Henceforth, we studied the pharmacokinetics and safety of dutasteride/tamsulosin 0.5 mg/0.4 mg capsules according to 76 polymorphisms in 17 candidate pharmacogenes. The study population comprised 79 healthy male volunteers enrolled in three bioequivalence, phase-I, crossover, open, randomized clinical trials with different study designs: the first was single dose in fed state, the second was a single dose in fasting state, and the third was a multiple dose. As key findings, CYP2D6 PMs (i.e., *4/*4 and *4/*5 subjects) and intermediate metabolizers (IMs) (i.e., *1/*4, *1/*5, *4/*15 individuals) presented higher AUC (p = 0.004), higher t1/2 (p = 0.008), and lower Cl/F (p = 0.006) when compared with NMs (*1/*1 individuals) and UMs (1/*1 × 2 individuals) after multiple testing correction. Moreover, fed volunteers showed significantly higher tmax than fasting individuals. Nominally significant associations were observed between dutasteride exposure and CYP3A4 and CYP3A5 genotype and between tamsulosin and ABCG2, CYP3A5, and SLC22A1 genotypes. No association between the occurrence of adverse drug reactions and genotype was observed. Nonetheless, higher incidence of adverse events was found in a multiple-dose clinical trial. Based on our results, we suggest that dose adjustments for PMs and UMs could be considered to ensure drug safety and effectiveness, respectively. Further studies are warranted to confirm other pharmacogenetic associations.