Plasma proteomic associations with genetics and health in the UK Biobank.

The Pharma Proteomics Project is a precompetitive biopharmaceutical consortium characterizing the plasma proteomic profiles of 54,219 UK Biobank participants. Here we provide a detailed summary of this initiative, including technical and biological validations, insights into proteomic disease signatures, and prediction modelling for various demographic and health indicators. We present comprehensive protein quantitative trait locus (pQTL) mapping of 2,923 proteins that identifies 14,287 primary genetic associations, of which 81% are previously undescribed, alongside ancestry-specific pQTL mapping in non-European individuals. The study provides an updated characterization of the genetic architecture of the plasma proteome, contextualized with projected pQTL discovery rates as sample sizes and proteomic assay coverages increase over time. We offer extensive insights into trans pQTLs across multiple biological domains, highlight genetic influences on ligand-receptor interactions and pathway perturbations across a diverse collection of cytokines and complement networks, and illustrate long-range epistatic effects of ABO blood group and FUT2 secretor status on proteins with gastrointestinal tissue-enriched expression. We demonstrate the utility of these data for drug discovery by extending the genetic proxied effects of protein targets, such as PCSK9, on additional endpoints, and disentangle specific genes and proteins perturbed at loci associated with COVID-19 susceptibility. This public-private partnership provides the scientific community with an open-access proteomics resource of considerable breadth and depth to help to elucidate the biological mechanisms underlying proteo-genomic discoveries and accelerate the development of biomarkers, predictive models and therapeutics1.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Sequence variants associating with urinary biomarkers.

Urine dipstick tests are widely used in routine medical care to diagnose kidney and urinary tract and metabolic diseases. Several environmental factors are known to affect the test results, whereas the effects of genetic diversity are largely unknown. We tested 32.5 million sequence variants for association with urinary biomarkers in a set of 150 274 Icelanders with urine dipstick measurements. We detected 20 association signals, of which 14 are novel, associating with at least one of five clinical entities defined by the urine dipstick: glucosuria, ketonuria, proteinuria, hematuria and urine pH. These include three independent glucosuria variants at SLC5A2, the gene encoding the sodium-dependent glucose transporter (SGLT2), a protein targeted pharmacologically to increase urinary glucose excretion in the treatment of diabetes. Two variants associating with proteinuria are in LRP2 and CUBN, encoding the co-transporters megalin and cubilin, respectively, that mediate proximal tubule protein uptake. One of the hematuria-associated variants is a rare, previously unreported 2.5 kb exonic deletion in COL4A3. Of the four signals associated with urine pH, we note that the pH-increasing alleles of two variants (POU2AF1, WDR72) associate significantly with increased risk of kidney stones. Our results reveal that genetic factors affect variability in urinary biomarkers, in both a disease dependent and independent context.

Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.

CpG islands and the regulation of transcription.

Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, CpG-rich, and predominantly nonmethylated. Most, perhaps all, CGIs are sites of transcription initiation, including thousands that are remote from currently annotated promoters. Shared DNA sequence features adapt CGIs for promoter function by destabilizing nucleosomes and attracting proteins that create a transcriptionally permissive chromatin state. Silencing of CGI promoters is achieved through dense CpG methylation or polycomb recruitment, again using their distinctive DNA sequence composition. CGIs are therefore generically equipped to influence local chromatin structure and simplify regulation of gene activity.

A unique DNA methylation signature defines a population of IFN-γ/IL-4 double-positive T cells during helminth infection.

Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4(+) T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN-γ/IL-4 double-positive cells. These IFN-γ(+) IL-4(+) cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN-γ(+) IL-4(-) (Th1) and IFN-γ(-) IL-4(+) (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN-γ(+) IL-4(+) cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co-existence of Th1 and Th2 characteristics.

Cell type-specific DNA methylation at intragenic CpG islands in the immune system.

Human and mouse genomes contain a similar number of CpG islands (CGIs), which are discrete CpG-rich DNA sequences associated with transcription start sites. In both species, ∼50% of all CGIs are remote from annotated promoters but, nevertheless, often have promoter-like features. To determine the role of CGI methylation in cell differentiation, we analyzed DNA methylation at a comprehensive CGI set in cells of the mouse hematopoietic lineage. Using a method that potentially detects ∼33% of genomic CpGs in the methylated state, we found that large differences in gene expression were accompanied by surprisingly few DNA methylation changes. There were, however, many DNA methylation differences between hematopoietic cells and a distantly related tissue, brain. Altered DNA methylation in the immune system occurred predominantly at CGIs within gene bodies, which have the properties of cell type-restricted promoters, but infrequently at annotated gene promoters or CGI flanking sequences (CGI "shores"). Unexpectedly, elevated intragenic CGI methylation correlated with silencing of the associated gene. Differentially methylated intragenic CGIs tended to lack H3K4me3 and associate with a transcriptionally repressive environment regardless of methylation state. Our results indicate that DNA methylation changes play a relatively minor role in the late stages of differentiation and suggest that intragenic CGIs represent regulatory sites of differential gene expression during the early stages of lineage specification.

Transcript-aware analysis of rare predicted loss-of-function variants in the UK Biobank elucidate new isoform-trait associations.

A single gene can produce multiple transcripts with distinct molecular functions. Rare-variant association tests often aggregate all coding variants across individual genes, without accounting for the variants' presence or consequence in resulting transcript isoforms. To evaluate the utility of transcript-aware variant sets, rare predicted loss-of-function (pLOF) variants were aggregated for 17,035 protein-coding genes using 55,558 distinct transcript-specific variant sets. These sets were tested for their association with 728 circulating proteins and 188 quantitative phenotypes across 406,921 individuals in the UK Biobank. The transcript-specific approach resulted in larger estimated effects of pLOF variants decreasing serum cis-protein levels compared to the gene-based approach (pbinom ≤ 2x10-16). Additionally, 251 quantitative trait associations were identified as being significant using the transcript-specific approach but not the gene-based approach, including PCSK5 transcript ENST00000376752 and standing height (transcript-specific statistic, P = 1.3x10-16, effect = 0.7 SD decrease; gene-based statistic, P = 0.02, effect = 0.05 SD decrease) and LDLR transcript ENST00000252444 and apolipoprotein B (transcript-specific statistic, P = 5.7x10-20, effect = 1.0 SD increase; gene-based statistic, P = 3.0x10-4, effect = 0.2 SD increase). This approach demonstrates the importance of considering the effect of pLOFs on specific transcript isoforms when performing rare-variant association studies.

Multiplexed Illumina sequencing libraries from picogram quantities of DNA.

BACKGROUND: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. RESULTS: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. CONCLUSIONS: Application of this method allows whole genome studies from samples where material or yields are limiting.

Using human genetics to improve safety assessment of therapeutics.

Human genetics research has discovered thousands of proteins associated with complex and rare diseases. Genome-wide association studies (GWAS) and studies of Mendelian disease have resulted in an increased understanding of the role of gene function and regulation in human conditions. Although the application of human genetics has been explored primarily as a method to identify potential drug targets and support their relevance to disease in humans, there is increasing interest in using genetic data to identify potential safety liabilities of modulating a given target. Human genetic variants can be used as a model to anticipate the effect of lifelong modulation of therapeutic targets and identify the potential risk for on-target adverse events. This approach is particularly useful for non-clinical safety evaluation of novel therapeutics that lack pharmacologically relevant animal models and can contribute to the intrinsic safety profile of a drug target. This Review illustrates applications of human genetics to safety studies during drug discovery and development, including assessing the potential for on- and off-target associated adverse events, carcinogenicity risk assessment, and guiding translational safety study designs and monitoring strategies. A summary of available human genetic resources and recommended best practices is provided. The challenges and future perspectives of translating human genetic information to identify risks for potential drug effects in preclinical and clinical development are discussed.

Advancing diagnosis and management of liver disease in adults through exome sequencing.

BACKGROUND: Whole-exome sequencing (WES) is an effective tool for diagnosis in patients who remain undiagnosed despite a comprehensive clinical work-up. While WES is being used increasingly in pediatrics and oncology, it remains underutilized in non-oncological adult medicine, including in patients with liver disease, in part based on the faulty premise that adults are unlikely to harbor rare genetic variants with large effect size. Here, we aim to assess the burden of rare genetic variants underlying liver disease in adults at two major tertiary referral academic medical centers. METHODS: WES analysis paired with comprehensive clinical evaluation was performed in fifty-two adult patients with liver disease of unknown etiology evaluated at two US tertiary academic health care centers. FINDINGS: Exome analysis uncovered a definitive or presumed diagnosis in 33% of patients (17/52) providing insight into their disease pathogenesis, with most of these patients (12/17) not having a known family history of liver disease. Our data shows that over two-thirds of undiagnosed liver disease patients attaining a genetic diagnosis were being evaluated for cholestasis or hepatic steatosis of unknown etiology. INTERPRETATION: This study reveals an underappreciated incidence and spectrum of genetic diseases presenting in adulthood and underscores the clinical value of incorporating exome sequencing in the evaluation and management of adults with liver disease of unknown etiology. FUNDING: S.V. is supported by the NIH/NIDDK (K08 DK113109 and R01 DK131033-01A1) and the Doris Duke Charitable Foundation Grant #2019081. This work was supported in part by NIH-funded Yale Liver Center, P30 DK34989.

Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins.

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.

Rare coding variants in DNA damage repair genes associated with timing of natural menopause.

The age of menopause is associated with fertility and disease risk, and its genetic control is of great interest. We use whole-exome sequences from 132,370 women in the UK Biobank to test for associations between rare damaging variants and age at natural menopause. Rare damaging variants in five genes are significantly associated with menopause: CHEK2 (p = 3.3 × 10-51), DCLRE1A (p = 8.4 × 10-13), and HELB (p = 5.7 × 10-7) with later menopause and TOP3A (p = 7.6 × 10-8) and CLPB (p = 8.1 × 10-7) with earlier menopause. Two additional genes are suggestive: RAD54L (p = 2.4 × 10-6) with later menopause and HROB (p = 2.9 × 10-6) with earlier menopause. In a follow-up analysis of repeated questionnaires in women who were initially premenopausal, CHEK2, TOP3A, and RAD54L genotypes are associated with subsequent menopause. Consistent with previous genome-wide association studies (GWASs), six of the seven genes are involved in the DNA damage repair pathway. Phenome-wide scans across 398,569 men and women revealed that in addition to known associations with cancers and blood cell counts, rare variants in CHEK2 are also associated with increased risk for uterine fibroids, polycystic ovary syndrome, and prostate hypertrophy; these associations are not shared with higher-penetrance breast cancer genes. Causal mediation analysis suggests that approximately 8% of the breast cancer risk conferred by CHEK2 pathogenic variants after menopause is mediated through delayed menopause.

Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity.

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease.

Gene-level analysis of rare variants in 379,066 whole exome sequences identifies an association of GIGYF1 loss of function with type 2 diabetes.

Sequencing of large cohorts offers an unprecedented opportunity to identify rare genetic variants and to find novel contributors to human disease. We used gene-based collapsing tests to identify genes associated with glucose, HbA1c and type 2 diabetes (T2D) diagnosis in 379,066 exome-sequenced participants in the UK Biobank. We identified associations for variants in GCK, HNF1A and PDX1, which are known to be involved in Mendelian forms of diabetes. Notably, we uncovered novel associations for GIGYF1, a gene not previously implicated by human genetics in diabetes. GIGYF1 predicted loss of function (pLOF) variants associated with increased levels of glucose (0.77 mmol/L increase, p = 4.42 × 10-12) and HbA1c (4.33 mmol/mol, p = 1.28 × 10-14) as well as T2D diagnosis (OR = 4.15, p = 6.14 × 10-11). Multiple rare variants contributed to these associations, including singleton variants. GIGYF1 pLOF also associated with decreased cholesterol levels as well as an increased risk of hypothyroidism. The association of GIGYF1 pLOF with T2D diagnosis replicated in an independent cohort from the Geisinger Health System. In addition, a common variant association for glucose and T2D was identified at the GIGYF1 locus. Our results highlight the role of GIGYF1 in regulating insulin signaling and protecting from diabetes.

GWAS of serum ALT and AST reveals an association of SLC30A10 Thr95Ile with hypermanganesemia symptoms.

Understanding mechanisms of hepatocellular damage may lead to new treatments for liver disease, and genome-wide association studies (GWAS) of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum activities have proven useful for investigating liver biology. Here we report 100 loci associating with both enzymes, using GWAS across 411,048 subjects in the UK Biobank. The rare missense variant SLC30A10 Thr95Ile (rs188273166) associates with the largest elevation of both enzymes, and this association replicates in the DiscovEHR study. SLC30A10 excretes manganese from the liver to the bile duct, and rare homozygous loss of function causes the syndrome hypermanganesemia with dystonia-1 (HMNDYT1) which involves cirrhosis. Consistent with hematological symptoms of hypermanganesemia, SLC30A10 Thr95Ile carriers have increased hematocrit and risk of iron deficiency anemia. Carriers also have increased risk of extrahepatic bile duct cancer. These results suggest that genetic variation in SLC30A10 adversely affects more individuals than patients with diagnosed HMNDYT1.

Association of the transthyretin variant V122I with polyneuropathy among individuals of African ancestry.

Hereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. V122I, a common pathogenic TTR mutation, is found in 3-4% of individuals of African ancestry in the United States and has been associated with cardiomyopathy and heart failure. To better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes in UK Biobank participants of African ancestry (n = 6062). Significant associations were tested for replication in the Penn Medicine Biobank (n = 5737) and the Million Veteran Program (n = 82,382). V122I was significantly associated with polyneuropathy in the UK Biobank (odds ratio [OR] = 6.4, 95% confidence interval [CI] 2.6-15.6, p = 4.2 × 10-5), which was replicated in the Penn Medicine Biobank (OR = 1.6, 95% CI 1.2-2.4, p = 6.0 × 10-3) and Million Veteran Program (OR = 1.5, 95% CI 1.2-1.8, p = 1.8 × 10-4). Polyneuropathy prevalence among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers compared with non-carriers (HR = 2.8, 95% CI 1.7-4.5, p = 2.6 × 10-5) in the UK Biobank, with 37.4% of V122I carriers having at least one of these manifestations by age 75. Our findings show that V122I carriers are at increased risk of polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR amyloidosis. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.

Specificity of Atonal and Scute bHLH factors: analysis of cognate E box binding sites and the influence of Senseless.

The question of how proneural bHLH transcription factors recognize and regulate their target genes is still relatively poorly understood. We previously showed that Scute (Sc) and Atonal (Ato) target genes have different cognate E box motifs, suggesting that specific DNA interactions contribute to differences in their target gene specificity. Here we show that Sc and Ato proteins (in combination with Daughterless) can activate reporter gene expression via their cognate E boxes in a non-neuronal cell culture system, suggesting that the proteins have strong intrinsic abilities to recognize different E box motifs in the absence of specialized cofactors. Functional comparison of E boxes from several target genes and site-directed mutagenesis of E box motifs suggests that specificity and activity require further sequence elements flanking both sides of the previously identified E box motifs. Moreover, the proneural cofactor, Senseless, can augment the function of Sc and Ato on their cognate E boxes and therefore may contribute to proneural specificity.

Phenotypes associated with genes encoding drug targets are predictive of clinical trial side effects.

Only a small fraction of early drug programs progress to the market, due to safety and efficacy failures, despite extensive efforts to predict safety. Characterizing the effect of natural variation in the genes encoding drug targets should present a powerful approach to predict side effects arising from drugging particular proteins. In this retrospective analysis, we report a correlation between the organ systems affected by genetic variation in drug targets and the organ systems in which side effects are observed. Across 1819 drugs and 21 phenotype categories analyzed, drug side effects are more likely to occur in organ systems where there is genetic evidence of a link between the drug target and a phenotype involving that organ system, compared to when there is no such genetic evidence (30.0 vs 19.2%; OR = 1.80). This result suggests that human genetic data should be used to predict safety issues associated with drug targets.

Author Correction: Phenotypes associated with genes encoding drug targets are predictive of clinical trial side effects.

In the original version of this article, there were errors in the labelling of the colours in the key of Figure 2, whereby the labeling of the third and fourth of the four colours was reversed. This has been corrected in both the PDF and HTML versions of the article.