Large-scale plasma proteomics comparisons through genetics and disease associations.

High-throughput proteomics platforms measuring thousands of proteins in plasma combined with genomic and phenotypic information have the power to bridge the gap between the genome and diseases. Here we performed association studies of Olink Explore 3072 data generated by the UK Biobank Pharma Proteomics Project1 on plasma samples from more than 50,000 UK Biobank participants with phenotypic and genotypic data, stratifying on British or Irish, African and South Asian ancestries. We compared the results with those of a SomaScan v4 study on plasma from 36,000 Icelandic people2, for 1,514 of whom Olink data were also available. We found modest correlation between the two platforms. Although cis protein quantitative trait loci were detected for a similar absolute number of assays on the two platforms (2,101 on Olink versus 2,120 on SomaScan), the proportion of assays with such supporting evidence for assay performance was higher on the Olink platform (72% versus 43%). A considerable number of proteins had genomic associations that differed between the platforms. We provide examples where differences between platforms may influence conclusions drawn from the integration of protein levels with the study of diseases. We demonstrate how leveraging the diverse ancestries of participants in the UK Biobank helps to detect novel associations and refine genomic location. Our results show the value of the information provided by the two most commonly used high-throughput proteomics platforms and demonstrate the differences between them that at times provides useful complementarity.

FLT3 stop mutation increases FLT3 ligand level and risk of autoimmune thyroid disease.

Autoimmune thyroid disease is the most common autoimmune disease and is highly heritable1. Here, by using a genome-wide association study of 30,234 cases and 725,172 controls from Iceland and the UK Biobank, we find 99 sequence variants at 93 loci, of which 84 variants are previously unreported2-7. A low-frequency (1.36%) intronic variant in FLT3 (rs76428106-C) has the largest effect on risk of autoimmune thyroid disease (odds ratio (OR) = 1.46, P = 2.37 × 10-24). rs76428106-C is also associated with systemic lupus erythematosus (OR = 1.90, P = 6.46 × 10-4), rheumatoid factor and/or anti-CCP-positive rheumatoid arthritis (OR = 1.41, P = 4.31 × 10-4) and coeliac disease (OR = 1.62, P = 1.20 × 10-4). FLT3 encodes fms-related tyrosine kinase 3, a receptor that regulates haematopoietic progenitor and dendritic cells. RNA sequencing revealed that rs76428106-C generates a cryptic splice site, which introduces a stop codon in 30% of transcripts that are predicted to encode a truncated protein, which lacks its tyrosine kinase domains. Each copy of rs76428106-C doubles the plasma levels of the FTL3 ligand. Activating somatic mutations in FLT3 are associated with acute myeloid leukaemia8 with a poor prognosis and rs76428106-C also predisposes individuals to acute myeloid leukaemia (OR = 1.90, P = 5.40 × 10-3). Thus, a predicted loss-of-function germline mutation in FLT3 causes a reduction in full-length FLT3, with a compensatory increase in the levels of its ligand and an increased disease risk, similar to that of a gain-of-function mutation.

Metabolic Profiling and Compound-Class Identification Reveal Alterations in Serum Triglyceride Levels in Mice Immunized with Human Vaccine Adjuvant Alum.

Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (MS) (liquid chromatography-MS)-based metabolic and lipid profiling to monitor the effects of the Alum adjuvant on mouse serum at 6, 24, 72, and 168 h post-vaccination. We propose a new strategy termed subclass identification and annotation for metabolomics for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long-chain unsaturated fatty acids (FAs) decreased at 24 h and TAGs with short-chain FAs decreased at 168 h. To our knowledge, this is the first report on the impact of human vaccine adjuvant Alum on the host metabolome, which may provide new insights into the mechanism of action of Alum.

C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates.

Introduction: C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults. Methods: Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF®01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses. Results: On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01- induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)-specific B cells to form HEL+ GC B cells in neonatal mice upon vaccination with HEL-OVA. Discussion: Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.

Genetics and epidemiology of mutational barcode-defined clonal hematopoiesis.

Clonal hematopoiesis (CH) arises when a substantial proportion of mature blood cells is derived from a single hematopoietic stem cell lineage. Using whole-genome sequencing of 45,510 Icelandic and 130,709 UK Biobank participants combined with a mutational barcode method, we identified 16,306 people with CH. Prevalence approaches 50% in elderly participants. Smoking demonstrates a dosage-dependent impact on risk of CH. CH associates with several smoking-related diseases. Contrary to published claims, we find no evidence that CH is associated with cardiovascular disease. We provide evidence that CH is driven by genes that are commonly mutated in myeloid neoplasia and implicate several new driver genes. The presence and nature of a driver mutation alters the risk profile for hematological disorders. Nevertheless, most CH cases have no known driver mutations. A CH genome-wide association study identified 25 loci, including 19 not implicated previously in CH. Splicing, protein and expression quantitative trait loci were identified for CD164 and TCL1A.

Sequence variant affects GCSAML splicing, mast cell specific proteins, and risk of urticaria.

Urticaria is a skin disorder characterized by outbreaks of raised pruritic wheals. In order to identify sequence variants associated with urticaria, we performed a meta-analysis of genome-wide association studies for urticaria with a total of 40,694 cases and 1,230,001 controls from Iceland, the UK, Finland, and Japan. We also performed transcriptome- and proteome-wide analyses in Iceland and the UK. We found nine sequence variants at nine loci associating with urticaria. The variants are at genes participating in type 2 immune responses and/or mast cell biology (CBLB, FCER1A, GCSAML, STAT6, TPSD1, ZFPM1), the innate immunity (C4), and NF-κB signaling. The most significant association was observed for the splice-donor variant rs56043070[A] (hg38: chr1:247556467) in GCSAML (MAF = 6.6%, OR = 1.24 (95%CI: 1.20-1.28), P-value = 3.6 × 10-44). We assessed the effects of the variants on transcripts, and levels of proteins relevant to urticaria pathophysiology. Our results emphasize the role of type 2 immune response and mast cell activation in the pathogenesis of urticaria. Our findings may point to an IgE-independent urticaria pathway that could help address unmet clinical need.

Physical and cognitive impact following SARS-CoV-2 infection in a large population-based case-control study.

BACKGROUND: Persistent symptoms are common after SARS-CoV-2 infection but correlation with objective measures is unclear. METHODS: We invited all 3098 adults who tested SARS-CoV-2 positive in Iceland before October 2020 to the deCODE Health Study. We compared multiple symptoms and physical measures between 1706 Icelanders with confirmed prior infection (cases) who participated, and 619 contemporary and 13,779 historical controls. Cases participated in the study 5-18 months after infection. RESULTS: Here we report that 41 of 88 symptoms are associated with prior infection, most significantly disturbed smell and taste, memory disturbance, and dyspnea. Measured objectively, cases had poorer smell and taste results, less grip strength, and poorer memory recall. Differences in grip strength and memory recall were small. No other objective measure associated with prior infection including heart rate, blood pressure, postural orthostatic tachycardia, oxygen saturation, exercise tolerance, hearing, and traditional inflammatory, cardiac, liver, and kidney blood biomarkers. There was no evidence of more anxiety or depression among cases. We estimate the prevalence of long Covid to be 7% at a median of 8 months after infection. CONCLUSIONS: We confirm that diverse symptoms are common months after SARS-CoV-2 infection but find few differences between cases and controls in objective parameters measured. These discrepancies between symptoms and physical measures suggest a more complicated contribution to symptoms related to prior infection than is captured with conventional tests. Traditional clinical assessment is not expected to be particularly informative in relating symptoms to a past SARS-CoV-2 infection.

Persistent symptoms are commonly reported after SARS-CoV-2 infection, and this is often described as long Covid. We compared different symptoms reported following SARS-CoV- 2 infection with the results obtained during various medical evaluations that are often used to assess health, such as blood tests, smell tests, taste tests, hearing tests, etc. We compared symptoms and test results between 1,706 Icelanders who had been infected previously with SARS-CoV-2 infection (cases) and 14,398 individuals who had not been infected (controls). Out of 88 assessed symptoms, 41 were more common in cases than controls. However, relatively few differences were seen in the results obtained from the various medical evaluations (cases had poorer smell and taste test results, slightly less grip strength, and slightly poorer memory recall than controls). The differences seen between symptoms and results of medical evaluations suggests that conventional clinical tests may not be informative in relating symptoms to a past SARS-CoV-2 infection.

Deficit of homozygosity among 1.52 million individuals and genetic causes of recessive lethality.

Genotypes causing pregnancy loss and perinatal mortality are depleted among living individuals and are therefore difficult to find. To explore genetic causes of recessive lethality, we searched for sequence variants with deficit of homozygosity among 1.52 million individuals from six European populations. In this study, we identified 25 genes harboring protein-altering sequence variants with a strong deficit of homozygosity (10% or less of predicted homozygotes). Sequence variants in 12 of the genes cause Mendelian disease under a recessive mode of inheritance, two under a dominant mode, but variants in the remaining 11 have not been reported to cause disease. Sequence variants with a strong deficit of homozygosity are over-represented among genes essential for growth of human cell lines and genes orthologous to mouse genes known to affect viability. The function of these genes gives insight into the genetics of intrauterine lethality. We also identified 1077 genes with homozygous predicted loss-of-function genotypes not previously described, bringing the total set of genes completely knocked out in humans to 4785.

Novel protein-based pneumococcal vaccines administered with the Th1-promoting adjuvant IC31 induce protective immunity against pneumococcal disease in neonatal mice.

Streptococcus pneumoniae is responsible for many vaccine-preventable deaths, annually causing around 1 million deaths in children younger than 5 years of age. A new generation of pneumococcal vaccines based on conserved proteins is being developed. We evaluated the immunogenicities and protective efficacies of four pneumococcal protein vaccine candidates, PcsB, StkP, PsaA, and PspA, in a neonatal mouse model. Mice were immunized three times and challenged intranasally with virulent pneumococci. All four proteins were immunogenic in neonatal mice, and antibody (Ab) responses were significantly enhanced by the novel adjuvant IC31, which consists of an antibacterial peptide (KLKL5KLK) and a synthetic oligodeoxynucleotide, ODN1a, that signals through Toll-like receptor 9 (TLR9). Two single proteins, StkP and PspA, combined with IC31 significantly reduced pneumococcal bacteremia but had no effects on lung infection. Three proteins, PcsB, StkP, and PsaA, were evaluated with alum or IC31. IC31 enhanced Ab responses and avidity to all three proteins, whereas alum enhanced Ab responses and avidity to StkP and PsaA only. Mice receiving the trivalent protein formulation with IC31 had significantly reduced bacteremia and lung infection compared to unvaccinated mice, but the level of protection was dependent on the dose of IC31. When PspA was added to the trivalent protein formulation, the dose of IC31 needed to obtain protective immunity could be reduced. These results demonstrate that a novel pneumococcal protein-based vaccine is immunogenic at an early age of mice and emphasize the benefits of using a combination of conserved proteins and an effective adjuvant to elicit potent protective immunity against invasive pneumococcal disease.

Effect of booster vaccination against Delta and Omicron SARS-CoV-2 variants in Iceland.

By the end of July 2021, the majority of the Icelandic population had received vaccination against COVID-19. In mid-July a wave of SARS-CoV-2 infections, dominated by the Delta variant, spread through the population, followed by an Omicron wave in December. A booster vaccination campaign was initiated to curb the spread of the virus. We estimate the risk of infection for different vaccine combinations using vaccination data from 276,028 persons and 963,557 qPCR tests for 277,687 persons. We measure anti-Spike-RBD antibody levels and ACE2-Spike binding inhibitory activity in 371 persons who received one of four recommended vaccination schedules with or without an mRNA vaccine booster. Overall, we find different antibody levels and inhibitory activity in recommended vaccination schedules, reflected in the observed risk of SARS-CoV-2 infections. We observe an increased protection following mRNA boosters, against both Omicron and Delta variant infections, although BNT162b2 boosters provide greater protection against Omicron than mRNA-1273 boosters.

Functional dissection of inherited non-coding variation influencing multiple myeloma risk.

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

A genome-wide meta-analysis identifies 50 genetic loci associated with carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy and has a largely unknown underlying biology. In a genome-wide association study of CTS (48,843 cases and 1,190,837 controls), we found 53 sequence variants at 50 loci associated with the syndrome. The most significant association is with a missense variant (p.Glu366Lys) in SERPINA1 that protects against CTS (P = 2.9 × 10-24, OR = 0.76). Through various functional analyses, we conclude that at least 22 genes mediate CTS risk and highlight the role of 19 CTS variants in the biology of the extracellular matrix. We show that the genetic component to the risk is higher in bilateral/recurrent/persistent cases than nonrecurrent/nonpersistent cases. Anthropometric traits including height and BMI are genetically correlated with CTS, in addition to early hormonal-replacement therapy, osteoarthritis, and restlessness. Our findings suggest that the components of the extracellular matrix play a key role in the pathogenesis of CTS.

HLA alleles, disease severity, and age associate with T-cell responses following infection with SARS-CoV-2.

Memory T-cell responses following SARS-CoV-2 infection have been extensively investigated but many studies have been small with a limited range of disease severity. Here we analyze SARS-CoV-2 reactive T-cell responses in 768 convalescent SARS-CoV-2-infected (cases) and 500 uninfected (controls) Icelanders. The T-cell responses are stable three to eight months after SARS-CoV-2 infection, irrespective of disease severity and even those with the mildest symptoms induce broad and persistent T-cell responses. Robust CD4+ T-cell responses are detected against all measured proteins (M, N, S and S1) while the N protein induces strongest CD8+ T-cell responses. CD4+ T-cell responses correlate with disease severity, humoral responses and age, whereas CD8+ T-cell responses correlate with age and functional antibodies. Further, CD8+ T-cell responses associate with several class I HLA alleles. Our results, provide new insight into HLA restriction of CD8+ T-cell immunity and other factors contributing to heterogeneity of T-cell responses following SARS-CoV-2 infection.

Genetic Associations and Architecture of Asthma-COPD Overlap.

BACKGROUND: Some people have characteristics of both asthma and COPD (asthma-COPD overlap), and evidence suggests they experience worse outcomes than those with either condition alone. RESEARCH QUESTION: What is the genetic architecture of asthma-COPD overlap, and do the determinants of risk for asthma-COPD overlap differ from those for COPD or asthma? STUDY DESIGN AND METHODS: We conducted a genome-wide association study in 8,068 asthma-COPD overlap case subjects and 40,360 control subjects without asthma or COPD of European ancestry in UK Biobank (stage 1). We followed up promising signals (P < 5 × 10-6) that remained associated in analyses comparing (1) asthma-COPD overlap vs asthma-only control subjects, and (2) asthma-COPD overlap vs COPD-only control subjects. These variants were analyzed in 12 independent cohorts (stage 2). RESULTS: We selected 31 independent variants for further investigation in stage 2, and discovered eight novel signals (P < 5 × 10-8) for asthma-COPD overlap (meta-analysis of stage 1 and 2 studies). These signals suggest a spectrum of shared genetic influences, some predominantly influencing asthma (FAM105A, GLB1, PHB, TSLP), others predominantly influencing fixed airflow obstruction (IL17RD, C5orf56, HLA-DQB1). One intergenic signal on chromosome 5 had not been previously associated with asthma, COPD, or lung function. Subgroup analyses suggested that associations at these eight signals were not driven by smoking or age at asthma diagnosis, and in phenome-wide scans, eosinophil counts, atopy, and asthma traits were prominent. INTERPRETATION: We identified eight signals for asthma-COPD overlap, which may represent loci that predispose to type 2 inflammation, and serious long-term consequences of asthma.

Large-scale integration of the plasma proteome with genetics and disease.

The plasma proteome can help bridge the gap between the genome and diseases. Here we describe genome-wide association studies (GWASs) of plasma protein levels measured with 4,907 aptamers in 35,559 Icelanders. We found 18,084 associations between sequence variants and levels of proteins in plasma (protein quantitative trait loci; pQTL), of which 19% were with rare variants (minor allele frequency (MAF) < 1%). We tested plasma protein levels for association with 373 diseases and other traits and identified 257,490 associations. We integrated pQTL and genetic associations with diseases and other traits and found that 12% of 45,334 lead associations in the GWAS Catalog are with variants in high linkage disequilibrium with pQTL. We identified 938 genes encoding potential drug targets with variants that influence levels of possible biomarkers. Combining proteomics, genomics and transcriptomics, we provide a valuable resource that can be used to improve understanding of disease pathogenesis and to assist with drug discovery and development.

Eighty-eight variants highlight the role of T cell regulation and airway remodeling in asthma pathogenesis.

Asthma is one of the most common chronic diseases affecting both children and adults. We report a genome-wide association meta-analysis of 69,189 cases and 702,199 controls from Iceland and UK biobank. We find 88 asthma risk variants at 56 loci, 19 previously unreported, and evaluate their effect on other asthma and allergic phenotypes. Of special interest are two low frequency variants associated with protection against asthma; a missense variant in TNFRSF8 and 3' UTR variant in TGFBR1. Functional studies show that the TNFRSF8 variant reduces TNFRSF8 expression both on cell surface and in soluble form, acting as loss of function. eQTL analysis suggests that the TGFBR1 variant acts through gain of function and together with an intronic variant in a downstream gene, SMAD3, points to defective TGFßR1 signaling as one of the biological perturbations increasing asthma risk. Our results increase the number of asthma variants and implicate genes with known role in T cell regulation, inflammation and airway remodeling in asthma pathogenesis.

Genetic predisposition to hypertension is associated with preeclampsia in European and Central Asian women.

Preeclampsia is a serious complication of pregnancy, affecting both maternal and fetal health. In genome-wide association meta-analysis of European and Central Asian mothers, we identify sequence variants that associate with preeclampsia in the maternal genome at ZNF831/20q13 and FTO/16q12. These are previously established variants for blood pressure (BP) and the FTO variant has also been associated with body mass index (BMI). Further analysis of BP variants establishes that variants at MECOM/3q26, FGF5/4q21 and SH2B3/12q24 also associate with preeclampsia through the maternal genome. We further show that a polygenic risk score for hypertension associates with preeclampsia. However, comparison with gestational hypertension indicates that additional factors modify the risk of preeclampsia.

A loss-of-function variant in ALOX15 protects against nasal polyps and chronic rhinosinusitis.

Nasal polyps (NP) are lesions on the nasal and paranasal sinus mucosa and are a risk factor for chronic rhinosinusitis (CRS). We performed genome-wide association studies on NP and CRS in Iceland and the UK (using UK Biobank data) with 4,366 NP cases, 5,608 CRS cases, and >700,000 controls. We found 10 markers associated with NP and 2 with CRS. We also tested 210 markers reported to associate with eosinophil count, yielding 17 additional NP associations. Of the 27 NP signals, 7 associate with CRS and 13 with asthma. Most notably, a missense variant in ALOX15 that causes a p.Thr560Met alteration in arachidonate 15-lipoxygenase (15-LO) confers large genome-wide significant protection against NP (P = 8.0 × 10-27, odds ratio = 0.32; 95% confidence interval = 0.26, 0.39) and CRS (P = 1.1 × 10-8, odds ratio = 0.64; 95% confidence interval = 0.55, 0.75). p.Thr560Met, carried by around 1 in 20 Europeans, was previously shown to cause near total loss of 15-LO enzymatic activity. Our findings identify 15-LO as a potential target for therapeutic intervention in NP and CRS.

Molecular Signatures of a TLR4 Agonist-Adjuvanted HIV-1 Vaccine Candidate in Humans.

Systems biology approaches have recently provided new insights into the mechanisms of action of human vaccines and adjuvants. Here, we investigated early transcriptional signatures induced in whole blood of healthy subjects following vaccination with a recombinant HIV-1 envelope glycoprotein subunit CN54gp140 adjuvanted with the TLR4 agonist glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) and correlated signatures to CN54gp140-specific serum antibody responses. Fourteen healthy volunteers aged 18-45 years were immunized intramuscularly three times at 1-month intervals and whole blood samples were collected at baseline, 6 h, and 1, 3, and 7 days post first immunization. Subtle changes in the transcriptomic profiles were observed following immunization, ranging from over 300 differentially expressed genes (DEGs) at day 1 to nearly 100 DEGs at day 7 following immunization. Functional pathway analysis revealed blood transcription modules (BTMs) related to general cell cycle activation, and innate immune cell activation at early time points, as well as BTMs related to T cells and B cell activation at the later time points post-immunization. Diverse CN54gp140-specific serum antibody responses of the subjects enabled their categorization into high or low responders, at early (<1 month) and late (up to 6 months) time points post vaccination. BTM analyses revealed repression of modules enriched in NK cells, and the mitochondrial electron chain, in individuals with high or sustained antigen-specific antibody responses. However, low responders showed an enhancement of BTMs associated with enrichment in myeloid cells and monocytes as well as integrin cell surface interactions. Flow cytometry analysis of peripheral blood mononuclear cells obtained from the subjects revealed an enhanced frequency of CD56dim NK cells in the majority of vaccines 14 days after vaccination as compared with the baseline. These results emphasize the utility of a systems biology approach to enhance our understanding on the mechanisms of action of TLR4 adjuvanted human vaccines.