Gene-based association study of rare variants in children of diverse ancestries implicates TNFRSF21 in the development of allergic asthma.

BACKGROUND: Most genetic studies of asthma and allergy have focused on common variation in individuals primarily of European ancestry. Studying the role of rare variation in quantitative phenotypes and in asthma phenotypes in populations of diverse ancestries can provide additional, important insights into the development of these traits. OBJECTIVE: We sought to examine the contribution of rare variants to different asthma- or allergy-associated quantitative traits in children with diverse ancestries and explore their role in asthma phenotypes. METHODS: We examined whole-genome sequencing data from children participants in longitudinal studies of asthma (n = 1035; parent-identified as 67% Black and 25% Hispanic) to identify rare variants (minor allele frequency < 0.01). We assigned variants to genes and tested for associations using an omnibus variant-set test between each of 24,902 genes and 8 asthma-associated quantitative traits. On combining our results with external data on predicted gene expression in humans and mouse knockout studies, we identified 3 candidate genes. A burden of rare variants in each gene and in a combined 3-gene score was tested for its associations with clinical phenotypes of asthma. Finally, published single-cell gene expression data in lower airway mucosal cells after allergen challenge were used to assess transcriptional responses to allergen. RESULTS: Rare variants in USF1 were significantly associated with blood neutrophil count (P = 2.18 × 10-7); rare variants in TNFRSF21 with total IgE (P = 6.47 × 10-6) and PIK3R6 with eosinophil count (P = 4.10 × 10-5) reached suggestive significance. These 3 findings were supported by independent data from human and mouse studies. A burden of rare variants in TNFRSF21 and in a 3-gene score was associated with allergy-related phenotypes in cohorts of children with mild and severe asthma. Furthermore, TNFRSF21 was significantly upregulated in bronchial basal epithelial cells from adults with allergic asthma but not in adults with allergies (but not asthma) after allergen challenge. CONCLUSIONS: We report novel associations between rare variants in genes and allergic and inflammatory phenotypes in children with diverse ancestries, highlighting TNFRSF21 as contributing to the development of allergic asthma.

Revealing polygenic pleiotropy using genetic risk scores for asthma.

In this study we examined how genetic risk for asthma associates with different features of the disease and with other medical conditions and traits. Using summary statistics from two multi-ancestry genome-wide association studies of asthma, we modeled polygenic risk scores (PRSs) and validated their predictive performance in the UK Biobank. We then performed phenome-wide association studies of the asthma PRSs with 371 heritable traits in the UK Biobank. We identified 228 total significant associations across a variety of organ systems, including associations that varied by PRS model, sex, age of asthma onset, ancestry, and human leukocyte antigen region alleles. Our results highlight pervasive pleiotropy between asthma and numerous other traits and conditions and elucidate pathways that contribute to asthma and its comorbidities.

Multi-omic association study identifies DNA methylation-mediated genotype and smoking exposure effects on lung function in children living in urban settings.

Impaired lung function in early life is associated with the subsequent development of chronic respiratory disease. Most genetic associations with lung function have been identified in adults of European descent and therefore may not represent those most relevant to pediatric populations and populations of different ancestries. In this study, we performed genome-wide association analyses of lung function in a multiethnic cohort of children (n = 1,035) living in low-income urban neighborhoods. We identified one novel locus at the TDRD9 gene in chromosome 14q32.33 associated with percent predicted forced expiratory volume in one second (FEV1) (p = 2.4x10-9; ßz = -0.31, 95% CI = -0.41- -0.21). Mendelian randomization and mediation analyses revealed that this genetic effect on FEV1 was partially mediated by DNA methylation levels at this locus in airway epithelial cells, which were also associated with environmental tobacco smoke exposure (p = 0.015). Promoter-enhancer interactions in airway epithelial cells revealed chromatin interaction loops between FEV1-associated variants in TDRD9 and the promoter region of the PPP1R13B gene, a stimulator of p53-mediated apoptosis. Expression of PPP1R13B in airway epithelial cells was significantly associated the FEV1 risk alleles (p = 1.3x10-5; ß = 0.12, 95% CI = 0.06-0.17). These combined results highlight a potential novel mechanism for reduced lung function in urban youth resulting from both genetics and smoking exposure.

African-specific alleles modify risk for asthma at the 17q12-q21 locus in African Americans.

BACKGROUND: Asthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry. METHODS: We tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American-specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults. RESULTS: Our studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma. CONCLUSIONS: Previously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases.

Fine-mapping studies distinguish genetic risks for childhood- and adult-onset asthma in the HLA region.

BACKGROUND: Genome-wide association studies of asthma have revealed robust associations with variation across the human leukocyte antigen (HLA) complex with independent associations in the HLA class I and class II regions for both childhood-onset asthma (COA) and adult-onset asthma (AOA). However, the specific variants and genes contributing to risk are unknown. METHODS: We used Bayesian approaches to perform genetic fine-mapping for COA and AOA (n=9432 and 21,556, respectively; n=318,167 shared controls) in White British individuals from the UK Biobank and to perform expression quantitative trait locus (eQTL) fine-mapping in immune (lymphoblastoid cell lines, n=398; peripheral blood mononuclear cells, n=132) and airway (nasal epithelial cells, n=188) cells from ethnically diverse individuals. We also examined putatively causal protein coding variation from protein crystal structures and conducted replication studies in independent multi-ethnic cohorts from the UK Biobank (COA n=1686; AOA n=3666; controls n=56,063). RESULTS: Genetic fine-mapping revealed both shared and distinct causal variation between COA and AOA in the class I region but only distinct causal variation in the class II region. Both gene expression levels and amino acid variation contributed to risk. Our results from eQTL fine-mapping and amino acid visualization suggested that the HLA-DQA1*03:01 allele and variation associated with expression of the nonclassical HLA-DQA2 and HLA-DQB2 genes accounted entirely for the most significant association with AOA in GWAS. Our studies also suggested a potentially prominent role for HLA-C protein coding variation in the class I region in COA. We replicated putatively causal variant associations in a multi-ethnic cohort. CONCLUSIONS: We highlight roles for both gene expression and protein coding variation in asthma risk and identified putatively causal variation and genes in the HLA region. A convergence of genomic, transcriptional, and protein coding evidence implicates the HLA-DQA2 and HLA-DQB2 genes and HLA-DQA1*03:01 allele in AOA.

Deconstructing a Syndrome: Genomic Insights Into PCOS Causal Mechanisms and Classification.

Polycystic ovary syndrome (PCOS) is among the most common disorders in women of reproductive age, affecting up to 15% worldwide, depending on the diagnostic criteria. PCOS is characterized by a constellation of interrelated reproductive abnormalities, including disordered gonadotropin secretion, increased androgen production, chronic anovulation, and polycystic ovarian morphology. It is frequently associated with insulin resistance and obesity. These reproductive and metabolic derangements cause major morbidities across the lifespan, including anovulatory infertility and type 2 diabetes (T2D). Despite decades of investigative effort, the etiology of PCOS remains unknown. Familial clustering of PCOS cases has indicated a genetic contribution to PCOS. There are rare Mendelian forms of PCOS associated with extreme phenotypes, but PCOS typically follows a non-Mendelian pattern of inheritance consistent with a complex genetic architecture, analogous to T2D and obesity, that reflects the interaction of susceptibility genes and environmental factors. Genomic studies of PCOS have provided important insights into disease pathways and have indicated that current diagnostic criteria do not capture underlying differences in biology associated with different forms of PCOS. We provide a state-of-the-science review of genetic analyses of PCOS, including an overview of genomic methodologies aimed at a general audience of non-geneticists and clinicians. Applications in PCOS will be discussed, including strengths and limitations of each study. The contributions of environmental factors, including developmental origins, will be reviewed. Insights into the pathogenesis and genetic architecture of PCOS will be summarized. Future directions for PCOS genetic studies will be outlined.

Inducible expression quantitative trait locus analysis of the MUC5AC gene in asthma in urban populations of children.

BACKGROUND: Mucus plugging can worsen asthma control, lead to reduced lung function and fatal exacerbations. MUC5AC is the secretory mucin implicated in mucus plugging, and MUC5AC gene expression has been associated with development of airway obstruction and asthma exacerbations in urban children with asthma. However, the genetic determinants of MUC5AC expression are not established. OBJECTIVES: This study sought to assess single-nucleotide polymorphisms (SNPs) that influence MUC5AC expression and relate to pulmonary functions in childhood asthma. METHODS: This study used RNA-sequencing data from upper airway samples and performed cis-expression quantitative trait loci (eQTL) and allele-specific expression analyses in 2 cohorts of predominantly Black and Hispanic urban children, a high asthma-risk birth cohort, and an exacerbation-prone asthma cohort. Inducible MUC5AC eQTLs were further investigated during incipient asthma exacerbations. Significant eQTLs SNPs were tested for associations with lung function measurements and their functional consequences were investigated in DNA regulatory databases. RESULTS: Two independent groups of SNPs in the MUC5AC gene that were significantly associated with MUC5AC expression were identified. Moreover, these SNPs showed stronger eQTL associations with MUC5AC expression during asthma exacerbations, which is consistent with inducible expression. SNPs in 1 group also showed significant association with decreased pulmonary functions. These SNPs included multiple EGR1 transcription factor binding sites, suggesting a mechanism of effect. CONCLUSIONS: These findings demonstrate the applicability of organ-specific RNA-sequencing data to determine genetic factors contributing to a key disease pathway. Specifically, they suggest important genetic variations that may underlie propensity to mucus plugging in asthma and could be important in targeted asthma phenotyping and disease management strategies.

The influence of proton pump inhibitor therapy on the outcome of infliximab therapy in inflammatory bowel disease: a patient-level meta-analysis of randomised controlled studies.

OBJECTIVE: In treating patients with inflammatory bowel disease (IBD), how concomitant medications influence the response to infliximab is largely unexplored. We aim to evaluate whether proton pump inhibitors (PPIs) affect the response to infliximab therapy in patients with IBD. DESIGN: Patient-level data of adult patients with moderate-to-severe IBD treated with infliximab were obtained from the Yale Open Data Access Framework. Multivariable analysis and propensity score-matched analysis were performed to assess week 30 remission rates, week 54 remission rates and hospitalisation rates in patients on infliximab therapy with and without PPI exposure. RESULTS: Among the five randomised controlled studies, there were 147 and 889 patients on infliximab with and without PPI therapy, respectively. Patients on PPI were older, more likely to be Caucasian and were less likely to be on immunomodulator therapy. Patients on PPI were significantly less likely to achieve week 30 remission on multivariable analysis (OR 0.45, p<0.001). Following propensity score matching adjusting for baseline difference in patient characteristics, the week 30 remission rates were 30% and 49% in patients with and without PPI therapy, respectively (p<0.001). Analysing separately for disease, the findings remained statistically significant in Crohn's disease but did not reach significance in UC. Similar results were seen with week 54 remission rates. Patients on PPI were also more likely to be hospitalised (15% vs 8%, p=0.007). Rates of adverse events such as gastroenteritis were not different between the two groups. CONCLUSION: In this patient-level meta-analysis of randomised controlled studies, we found that patients with IBD taking PPI were less likely to achieve remission while on infliximab therapy. The results of our study warrant further investigation into the effect of PPI on IBD outcomes and therapies.

Distinct subtypes of polycystic ovary syndrome with novel genetic associations: An unsupervised, phenotypic clustering analysis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common, complex genetic disorder affecting up to 15% of reproductive-age women worldwide, depending on the diagnostic criteria applied. These diagnostic criteria are based on expert opinion and have been the subject of considerable controversy. The phenotypic variation observed in PCOS is suggestive of an underlying genetic heterogeneity, but a recent meta-analysis of European ancestry PCOS cases found that the genetic architecture of PCOS defined by different diagnostic criteria was generally similar, suggesting that the criteria do not identify biologically distinct disease subtypes. We performed this study to test the hypothesis that there are biologically relevant subtypes of PCOS. METHODS AND FINDINGS: Using biochemical and genotype data from a previously published PCOS genome-wide association study (GWAS), we investigated whether there were reproducible phenotypic subtypes of PCOS with subtype-specific genetic associations. Unsupervised hierarchical cluster analysis was performed on quantitative anthropometric, reproductive, and metabolic traits in a genotyped cohort of 893 PCOS cases (median and interquartile range [IQR]: age = 28 [25-32], body mass index [BMI] = 35.4 [28.2-41.5]). The clusters were replicated in an independent, ungenotyped cohort of 263 PCOS cases (median and IQR: age = 28 [24-33], BMI = 35.7 [28.4-42.3]). The clustering revealed 2 distinct PCOS subtypes: a "reproductive" group (21%-23%), characterized by higher luteinizing hormone (LH) and sex hormone binding globulin (SHBG) levels with relatively low BMI and insulin levels, and a "metabolic" group (37%-39%), characterized by higher BMI, glucose, and insulin levels with lower SHBG and LH levels. We performed a GWAS on the genotyped cohort, limiting the cases to either the reproductive or metabolic subtypes. We identified alleles in 4 loci that were associated with the reproductive subtype at genome-wide significance (PRDM2/KAZN, P = 2.2 × 10-10; IQCA1, P = 2.8 × 10-9; BMPR1B/UNC5C, P = 9.7 × 10-9; CDH10, P = 1.2 × 10-8) and one locus that was significantly associated with the metabolic subtype (KCNH7/FIGN, P = 1.0 × 10-8). We developed a predictive model to classify a separate, family-based cohort of 73 women with PCOS (median and IQR: age = 28 [25-33], BMI = 34.3 [27.8-42.3]) and found that the subtypes tended to cluster in families and that carriers of previously reported rare variants in DENND1A, a gene that regulates androgen biosynthesis, were significantly more likely to have the reproductive subtype of PCOS. Limitations of our study were that only PCOS cases of European ancestry diagnosed by National Institutes of Health (NIH) criteria were included, the sample sizes for the subtype GWAS were small, and the GWAS findings were not replicated. CONCLUSIONS: In conclusion, we have found reproducible reproductive and metabolic subtypes of PCOS. Furthermore, these subtypes were associated with novel, to our knowledge, susceptibility loci. Our results suggest that these subtypes are biologically relevant because they appear to have distinct genetic architecture. This study demonstrates how phenotypic subtyping can be used to gain additional insights from GWAS data.

The contribution of rare genetic variants to the pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a highly heritable disorder, but only a small proportion of the heritability can be accounted for by common genetic risk variants identified to date. It is possible that variants with lower allele frequencies that cannot be detected using genome-wide association study arrays contribute to PCOS. Here, we discuss the challenges inherent to studying rare genetic variants in complex disease and review several recent studies that have used DNA sequencing techniques to investigate whether rare variants play a role in PCOS pathogenesis. We evaluate these findings in the context of the latest literature in PCOS and complex disease genetics.

Hyperandrogenemia is Common in Asymptomatic Women and is Associated with Increased Metabolic Risk.

OBJECTIVE: Women with metabolic syndrome (MetS) have higher endogenous testosterone (T) levels than unaffected women. This study investigated whether hyperandrogenemia (HA) was a marker for increased cardiometabolic risk in reproductively normal premenopausal women. METHODS: Reproductive hormones and metabolic parameters were assessed in 198 women with regular menses and no clinical hyperandrogenism (eumenorrheic [EM]). Hyperandrogenic EM women were compared with 110 women with NIH criteria polycystic ovary syndrome. RESULTS: Twenty-two percent of EM women had HA. Levels of non-sex hormone-binding globulin (SHBG)-bound T were elevated in 68% of women, total T levels were elevated in 43% of women, and dehydroepiandrosterone sulfate levels were elevated in 30% of women. The prevalence of HA increased with BMI category (P = 0.01): 12% for BMI < 25 kg/m2 , 22% for BMI of 25 to 30 kg/m2 , and 31% for BMI ≥ 30 kg/m2 . MetS (adjusted odds ratio 2.9; 95% CI: 1.2-6.9) and dysglycemia risks (adjusted odds ratio 2.7; 95% CI: 1.2-5.8) were increased in hyperandrogenic EM women compared with normoandrogenic EM women, with adjustment for BMI. SHBG levels were independently associated with these metabolic end points (P < 0.001), whereas androgen levels were not. A cluster analysis confirmed that there was a discrete subset of EM women with HA and metabolic abnormalities. CONCLUSIONS: HA is common in EM women and is associated with increased risks for MetS and dysglycemia. However, low SHBG levels rather than elevated androgen levels may be the primary predictor of this relationship with metabolic dysfunction.

Family-Based Quantitative Trait Meta-Analysis Implicates Rare Noncoding Variants in DENND1A in Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is among the most common endocrine disorders of premenopausal women, affecting 5% to15% of this population depending on the diagnostic criteria applied. It is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is highly heritable, but only a small proportion of this heritability can be accounted for by the common genetic susceptibility variants identified to date. OBJECTIVE: The objective of this study was to test whether rare genetic variants contribute to PCOS pathogenesis. DESIGN, PATIENTS, AND METHODS: We performed whole-genome sequencing on DNA from 261 individuals from 62 families with one or more daughters with PCOS. We tested for associations of rare variants with PCOS and its concomitant hormonal traits using a quantitative trait meta-analysis. RESULTS: We found rare variants in DENND1A (P = 5.31 × 10-5, adjusted P = 0.039) that were significantly associated with reproductive and metabolic traits in PCOS families. CONCLUSIONS: Common variants in DENND1A have previously been associated with PCOS diagnosis in genome-wide association studies. Subsequent studies indicated that DENND1A is an important regulator of human ovarian androgen biosynthesis. Our findings provide additional evidence that DENND1A plays a central role in PCOS and suggest that rare noncoding variants contribute to disease pathogenesis.

Functional Genetic Variation in the Anti-Müllerian Hormone Pathway in Women With Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a highly heritable, common endocrine disorder characterized by hyperandrogenism, irregular menses, and polycystic ovaries. PCOS is often accompanied by elevated levels of anti-Müllerian hormone (AMH). AMH inhibits follicle maturation. AMH also inhibits steroidogenesis through transcriptional repression of CYP17A1. We recently identified 16 rare PCOS-specific pathogenic variants in AMH. OBJECTIVE: To test whether additional members of the AMH signaling pathway also contribute to the etiology of PCOS. PARTICIPANTS/DESIGN: Targeted resequencing of coding and regulatory regions of AMH and its specific type 2 receptor, AMHR2, was performed on 608 women affected with PCOS and 142 reproductively normal control women. Prediction tools of deleteriousness and in silico evidence of epigenetic modification were used to prioritize variants for functional evaluation. Dual-luciferase reporter assays and splicing assays were used to measure the impact of genetic variants on function. RESULTS: We identified 20 additional variants in/near AMH and AMHR2 with significantly reduced signaling activity in in vitro assays. Collectively, from our previous study and as reported herein, we have identified a total of 37 variants with impaired activity in/near AMH and AMHR2 in 41 women affected with PCOS, or 6.7% of our PCOS cohort. Furthermore, no functional variants were observed in the 142 phenotyped controls. The functional variants were significantly associated with PCOS in our cohort of 608 women with PCOS and 142 controls (P = 2.3 × 10-5) and very strongly associated with PCOS relative to a larger non-Finnish European (gnomAD) population-based control cohort (P < 1 × 10-9). CONCLUSION: The AMH signaling cascade plays an important role in PCOS etiology.

Comparative evaluation of isoform-level gene expression estimation algorithms for RNA-seq and exon-array platforms.

Given that the majority of multi-exon genes generate diverse functional products, it is important to evaluate expression at the isoform level. Previous studies have demonstrated strong gene-level correlations between RNA sequencing (RNA-seq) and microarray platforms, but have not studied their concordance at the isoform level. We performed transcript abundance estimation on raw RNA-seq and exon-array expression profiles available for common glioblastoma multiforme samples from The Cancer Genome Atlas using different analysis pipelines, and compared both the isoform- and gene-level expression estimates between programs and platforms. The results showed better concordance between RNA-seq/exon-array and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) platforms for fold change estimates than for raw abundance estimates, suggesting that fold change normalization against a control is an important step for integrating expression data across platforms. Based on RT-qPCR validations, eXpress and Multi-Mapping Bayesian Gene eXpression (MMBGX) programs achieved the best performance for RNA-seq and exon-array platforms, respectively, for deriving the isoform-level fold change values. While eXpress achieved the highest correlation with the RT-qPCR and exon-array (MMBGX) results overall, RSEM was more highly correlated with MMBGX for the subset of transcripts that are highly variable across the samples. eXpress appears to be most successful in discriminating lowly expressed transcripts, but IsoformEx and RSEM correlate more strongly with MMBGX for highly expressed transcripts. The results also reinforce how potentially important isoform-level expression changes can be masked by gene-level estimates, and demonstrate that exon arrays yield comparable results to RNA-seq for evaluating isoform-level expression changes.