Selection, optimization and validation of ten chronic disease polygenic risk scores for clinical implementation in diverse US populations.

Polygenic risk scores (PRSs) have improved in predictive performance, but several challenges remain to be addressed before PRSs can be implemented in the clinic, including reduced predictive performance of PRSs in diverse populations, and the interpretation and communication of genetic results to both providers and patients. To address these challenges, the National Human Genome Research Institute-funded Electronic Medical Records and Genomics (eMERGE) Network has developed a framework and pipeline for return of a PRS-based genome-informed risk assessment to 25,000 diverse adults and children as part of a clinical study. From an initial list of 23 conditions, ten were selected for implementation based on PRS performance, medical actionability and potential clinical utility, including cardiometabolic diseases and cancer. Standardized metrics were considered in the selection process, with additional consideration given to strength of evidence in African and Hispanic populations. We then developed a pipeline for clinical PRS implementation (score transfer to a clinical laboratory, validation and verification of score performance), and used genetic ancestry to calibrate PRS mean and variance, utilizing genetically diverse data from 13,475 participants of the All of Us Research Program cohort to train and test model parameters. Finally, we created a framework for regulatory compliance and developed a PRS clinical report for return to providers and for inclusion in an additional genome-informed risk assessment. The initial experience from eMERGE can inform the approach needed to implement PRS-based testing in diverse clinical settings.

Pediatric Features of Genetic Predisposition to Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) has historically been conceptualized as a disorder of the reproductive system in women. However, offspring of women with PCOS begin to show metabolic features of PCOS in childhood, suggestive of childhood manifestations. OBJECTIVE: To identify childhood manifestations of genetic risk for PCOS. METHODS: We calculated a PCOS polygenic risk score (PRS) for 12 350 girls and boys in 4 pediatric cohorts-ALSPAC (UK), COPSAC (Denmark), Project Viva (USA), and The HOLBÆK Study (Denmark). We tested for association of the PRS with PCOS-related phenotypes throughout childhood and with age at pubarche and age at peak height velocity and meta-analyzed effects across cohorts using fixed-effect models. RESULTS: Higher PRS for PCOS was associated with higher body mass index in midchildhood (0.05 kg/m2 increase per 1 SD of PRS, 95% CI 0.03, 0.07, P = 3 × 10-5) and higher risk of obesity in early childhood (OR 1.34, 95% CI 1.13, 1.59, P = .0009); both persisted through late adolescence (P all ≤.03). Higher PCOS PRS was associated with earlier age at pubarche (0.85-month decrease per 1 SD of PRS, 95% CI -1.44, -0.26, P = .005) and younger age at peak height velocity (0.64-month decrease per 1 SD of PRS, 95% CI -0.94, -0.33, P = 4 × 10-5). CONCLUSION: Genetic risk factors for PCOS are associated with alterations in metabolic, growth, and developmental traits in childhood. Thus, PCOS may not simply be a condition that affects women of reproductive age but, rather, a possible manifestation of an underlying condition that affects both sexes starting in early life.

Selection, optimization, and validation of ten chronic disease polygenic risk scores for clinical implementation in diverse populations.

Polygenic risk scores (PRS) have improved in predictive performance supporting their use in clinical practice. Reduced predictive performance of PRS in diverse populations can exacerbate existing health disparities. The NHGRI-funded eMERGE Network is returning a PRS-based genome-informed risk assessment to 25,000 diverse adults and children. We assessed PRS performance, medical actionability, and potential clinical utility for 23 conditions. Standardized metrics were considered in the selection process with additional consideration given to strength of evidence in African and Hispanic populations. Ten conditions were selected with a range of high-risk thresholds: atrial fibrillation, breast cancer, chronic kidney disease, coronary heart disease, hypercholesterolemia, prostate cancer, asthma, type 1 diabetes, obesity, and type 2 diabetes. We developed a pipeline for clinical PRS implementation, used genetic ancestry to calibrate PRS mean and variance, created a framework for regulatory compliance, and developed a PRS clinical report. eMERGE's experience informs the infrastructure needed to implement PRS-based implementation in diverse clinical settings.

Evidence From Men for Ovary-independent Effects of Genetic Risk Factors for Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is characterized by ovulatory dysfunction and hyperandrogenism and can be associated with cardiometabolic dysfunction, but it remains unclear which of these features are inciting causes and which are secondary consequences. OBJECTIVE: To determine whether ovarian function is necessary for genetic risk factors for PCOS to produce nonreproductive phenotypes. DESIGN, SETTING, AND PARTICIPANTS: Cohort of 176 360 men in the UK Biobank and replication cohort of 37 348 men in the Estonian Biobank. MAIN OUTCOME MEASURES: We calculated individual PCOS polygenic risk scores (PRS), tested for association of these PRS with PCOS-related phenotypes using linear and logistic regression and performed mediation analysis. RESULTS: For every 1 SD increase in the PCOS PRS, men had increased odds of obesity (odds ratio [OR]: 1.09; 95% CI, 1.08-1.10; P = 1 × 10-49), type 2 diabetes mellitus (T2DM) (OR: 1.08; 95% CI, 1.05-1.10; P = 3 × 10-12), coronary artery disease (CAD) (OR: 1.03; 95% CI, 1.01-1.04; P = 0.0029), and marked androgenic alopecia (OR: 1.03; 95% CI, 1.02-1.05; P = 3 × 10-5). Body mass index (BMI), hemoglobin A1c, triglycerides, and free androgen index increased as the PRS increased, whereas high-density lipoprotein cholesterol and SHBG decreased (all P < .0001). The association between the PRS and CAD appeared to be completely mediated by BMI, whereas the associations with T2DM and marked androgenic alopecia appeared to be partially mediated by BMI. CONCLUSIONS: Genetic risk factors for PCOS have phenotypic consequences in men, indicating that they can act independently of ovarian function. Thus, PCOS in women may not always be a primary disorder of the ovaries.

Blood Leukocyte DNA Methylation Predicts Risk of Future Myocardial Infarction and Coronary Heart Disease.

BACKGROUND: DNA methylation is implicated in coronary heart disease (CHD), but current evidence is based on small, cross-sectional studies. We examined blood DNA methylation in relation to incident CHD across multiple prospective cohorts. METHODS: Nine population-based cohorts from the United States and Europe profiled epigenome-wide blood leukocyte DNA methylation using the Illumina Infinium 450k microarray, and prospectively ascertained CHD events including coronary insufficiency/unstable angina, recognized myocardial infarction, coronary revascularization, and coronary death. Cohorts conducted race-specific analyses adjusted for age, sex, smoking, education, body mass index, blood cell type proportions, and technical variables. We conducted fixed-effect meta-analyses across cohorts. RESULTS: Among 11 461 individuals (mean age 64 years, 67% women, 35% African American) free of CHD at baseline, 1895 developed CHD during a mean follow-up of 11.2 years. Methylation levels at 52 CpG (cytosine-phosphate-guanine) sites were associated with incident CHD or myocardial infarction (false discovery rate<0.05). These CpGs map to genes with key roles in calcium regulation (ATP2B2, CASR, GUCA1B, HPCAL1), and genes identified in genome- and epigenome-wide studies of serum calcium (CASR), serum calcium-related risk of CHD (CASR), coronary artery calcified plaque (PTPRN2), and kidney function (CDH23, HPCAL1), among others. Mendelian randomization analyses supported a causal effect of DNA methylation on incident CHD; these CpGs map to active regulatory regions proximal to long non-coding RNA transcripts. CONCLUSION: Methylation of blood-derived DNA is associated with risk of future CHD across diverse populations and may serve as an informative tool for gaining further insight on the development of CHD.

The Consortium of Metabolomics Studies (COMETS): Metabolomics in 47 Prospective Cohort Studies.

The Consortium of Metabolomics Studies (COMETS) was established in 2014 to facilitate large-scale collaborative research on the human metabolome and its relationship with disease etiology, diagnosis, and prognosis. COMETS comprises 47 cohorts from Asia, Europe, North America, and South America that together include more than 136,000 participants with blood metabolomics data on samples collected from 1985 to 2017. Metabolomics data were provided by 17 different platforms, with the most frequently used labs being Metabolon, Inc. (14 cohorts), the Broad Institute (15 cohorts), and Nightingale Health (11 cohorts). Participants have been followed for a median of 23 years for health outcomes including death, cancer, cardiovascular disease, diabetes, and others; many of the studies are ongoing. Available exposure-related data include common clinical measurements and behavioral factors, as well as genome-wide genotype data. Two feasibility studies were conducted to evaluate the comparability of metabolomics platforms used by COMETS cohorts. The first study showed that the overlap between any 2 different laboratories ranged from 6 to 121 metabolites at 5 leading laboratories. The second study showed that the median Spearman correlation comparing 111 overlapping metabolites captured by Metabolon and the Broad Institute was 0.79 (interquartile range, 0.56-0.89).

Interrogation of human hematopoiesis at single-cell and single-variant resolution.

Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations.

Meta-analysis of genome-wide association studies for body fat distribution in 694 649 individuals of European ancestry.

More than one in three adults worldwide is either overweight or obese. Epidemiological studies indicate that the location and distribution of excess fat, rather than general adiposity, are more informative for predicting risk of obesity sequelae, including cardiometabolic disease and cancer. We performed a genome-wide association study meta-analysis of body fat distribution, measured by waist-to-hip ratio (WHR) adjusted for body mass index (WHRadjBMI), and identified 463 signals in 346 loci. Heritability and variant effects were generally stronger in women than men, and we found approximately one-third of all signals to be sexually dimorphic. The 5% of individuals carrying the most WHRadjBMI-increasing alleles were 1.62 times more likely than the bottom 5% to have a WHR above the thresholds used for metabolic syndrome. These data, made publicly available, will inform the biology of body fat distribution and its relationship with disease.

A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.

While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs.

A Conversation with Kurt and Rochelle Hirschhorn.

In this interview, Kurt and Rochelle Hirschhorn talk with their son, Joel, about their research and collaborations, the early years of medical genetics, the development of genetic counseling, the challenges of being a woman in science, and new challenges and directions for the study of human genetics.

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms.

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.

The Genetic Landscape of Renal Complications in Type 1 Diabetes.

Diabetes is the leading cause of ESRD. Despite evidence for a substantial heritability of diabetic kidney disease, efforts to identify genetic susceptibility variants have had limited success. We extended previous efforts in three dimensions, examining a more comprehensive set of genetic variants in larger numbers of subjects with type 1 diabetes characterized for a wider range of cross-sectional diabetic kidney disease phenotypes. In 2843 subjects, we estimated that the heritability of diabetic kidney disease was 35% (P=6.4×10-3). Genome-wide association analysis and replication in 12,540 individuals identified no single variants reaching stringent levels of significance and, despite excellent power, provided little independent confirmation of previously published associated variants. Whole-exome sequencing in 997 subjects failed to identify any large-effect coding alleles of lower frequency influencing the risk of diabetic kidney disease. However, sets of alleles increasing body mass index (P=2.2×10-5) and the risk of type 2 diabetes (P=6.1×10-4) associated with the risk of diabetic kidney disease. We also found genome-wide genetic correlation between diabetic kidney disease and failure at smoking cessation (P=1.1×10-4). Pathway analysis implicated ascorbate and aldarate metabolism (P=9.0×10-6), and pentose and glucuronate interconversions (P=3.0×10-6) in pathogenesis of diabetic kidney disease. These data provide further evidence for the role of genetic factors influencing diabetic kidney disease in those with type 1 diabetes and highlight some key pathways that may be responsible. Altogether these results reveal important biology behind the major cause of kidney disease.

Large-Scale Exome-wide Association Analysis Identifies Loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases.

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of âˆ¼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3' UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases.

Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells.

Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.

Whole exome sequencing identifies RAI1 mutation in a morbidly obese child diagnosed with ROHHAD syndrome.

CONTEXT: The current obesity epidemic is attributed to complex interactions between genetic and environmental factors. However, a limited number of cases, especially those with early-onset severe obesity, are linked to single gene defects. Rapid-onset obesity with hypothalamic dysfunction, hypoventilation and autonomic dysregulation (ROHHAD) is one of the syndromes that presents with abrupt-onset extreme weight gain with an unknown genetic basis. OBJECTIVE: To identify the underlying genetic etiology in a child with morbid early-onset obesity, hypoventilation, and autonomic and behavioral disturbances who was clinically diagnosed with ROHHAD syndrome. Design/Setting/Intervention: The index patient was evaluated at an academic medical center. Whole-exome sequencing was performed on the proband and his parents. Genetic variants were validated by Sanger sequencing. RESULTS: We identified a novel de novo nonsense mutation, c.3265 C>T (p.R1089X), in the retinoic acid-induced 1 (RAI1) gene in the proband. Mutations in the RAI1 gene are known to cause Smith-Magenis syndrome (SMS). On further evaluation, his clinical features were not typical of either SMS or ROHHAD syndrome. CONCLUSIONS: This study identifies a de novo RAI1 mutation in a child with morbid obesity and a clinical diagnosis of ROHHAD syndrome. Although extreme early-onset obesity, autonomic disturbances, and hypoventilation are present in ROHHAD, several of the clinical findings are consistent with SMS. This case highlights the challenges in the diagnosis of ROHHAD syndrome and its potential overlap with SMS. We also propose RAI1 as a candidate gene for children with morbid obesity.

Heterozygous mutations in natriuretic peptide receptor-B (NPR2) gene as a cause of short stature.

Based on the observation of reduced stature in relatives of patients with acromesomelic dysplasia, Maroteaux type (AMDM), caused by homozygous or compound heterozygous mutations in natriuretic peptide receptor-B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some cases of idiopathic short stature (ISS). We enrolled 192 unrelated patients with short stature and 192 controls of normal height and identified seven heterozygous NPR2 missense or splice site mutations all in the short stature patients, including one de novo splice site variant. Three of the six inherited variants segregated with short stature in the family. Nine additional rare nonsynonymous NPR2 variants were found in three additional cohorts. Functional studies identified eight loss-of-function mutations in short individuals and one gain-of-function mutation in tall individuals. With these data, we were able to rigorously verify that NPR2 functional haploinsufficiency contributes to short stature. We estimate a prevalence of NPR2 haploinsufficiency of between 0 and 1/26 in people with ISS. We suggest that NPR2 gain of function may be a more common cause of tall stature than previously recognized.

Gene expression analysis identifies global gene dosage sensitivity in cancer.

Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gene expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying this method to 16,172 patient-derived tumor samples, we replicated many loci with aberrant copy numbers and identified recurrently disrupted genes in genomically unstable cancers.

Genome-wide association analyses identify variants in developmental genes associated with hypospadias.

Hypospadias is a common congenital condition in boys in which the urethra opens on the underside of the penis. We performed a genome-wide association study on 1,006 surgery-confirmed hypospadias cases and 5,486 controls from Denmark. After replication genotyping of an additional 1,972 cases and 1,812 controls from Denmark, the Netherlands and Sweden, 18 genomic regions showed independent association with P < 5 × 10(-8). Together, these loci explain 9% of the liability to developing this condition. Several of the identified regions harbor genes with key roles in embryonic development (including HOXA4, IRX5, IRX6 and EYA1). Subsequent pathway analysis with GRAIL and DEPICT provided additional insight into possible genetic mechanisms causing hypospadias.

Short stature, accelerated bone maturation, and early growth cessation due to heterozygous aggrecan mutations.

CONTEXT: Many children with idiopathic short stature have a delayed bone age. Idiopathic short stature with advanced bone age is far less common. OBJECTIVE: The aim was to identify underlying genetic causes of short stature with advanced bone age. SETTING AND DESIGN: We used whole-exome sequencing to study three families with autosomal-dominant short stature, advanced bone age, and premature growth cessation. RESULTS: Affected individuals presented with short stature [adult heights -2.3 to -4.2 standard deviation scores (SDS)] with histories of early growth cessation or childhood short stature (height SDS -1.9 to -3.5 SDS), advancement of bone age, and normal endocrine evaluations. Whole-exome sequencing identified novel heterozygous variants in ACAN, which encodes aggrecan, a proteoglycan in the extracellular matrix of growth plate and other cartilaginous tissues. The variants were present in all affected, but in no unaffected, family members. In Family 1, a novel frameshift mutation in exon 3 (c.272delA) was identified, which is predicted to cause early truncation of the aggrecan protein. In Family 2, a base-pair substitution was found in a highly conserved location within a splice donor site (c.2026+1G>A), which is also likely to alter the amino acid sequence of a large portion of the protein. In Family 3, a missense variant (c.7064T>C) in exon 14 affects a highly conserved residue (L2355P) and is strongly predicted to perturb protein function. CONCLUSIONS: Our study demonstrates that heterozygous mutations in ACAN can cause a mild skeletal dysplasia, which presents clinically as short stature with advanced bone age. The accelerating effect on skeletal maturation has not previously been noted in the few prior reports of human ACAN mutations. Our findings thus expand the spectrum of ACAN defects and provide a new molecular genetic etiology for the unusual child who presents with short stature and accelerated skeletal maturation.

An excess of risk-increasing low-frequency variants can be a signal of polygenic inheritance in complex diseases.

In most complex diseases, much of the heritability remains unaccounted for by common variants. It has been postulated that lower-frequency variants contribute to the remaining heritability. Here, we describe a method to test for polygenic inheritance from lower-frequency variants by using GWAS summary association statistics. We explored scenarios with many causal low-frequency variants and showed that there is more power to detect risk variants than to detect protective variants, resulting in an increase in the ratio of detected risk to protective variants (R/P ratio). Such an excess can also occur if risk variants are present and kept at lower frequencies because of negative selection. The R/P ratio can be falsely elevated because of reasons unrelated to polygenic inheritance, such as uneven sample sizes or asymmetric population stratification, so precautions to correct for these confounders are essential. We tested our method on published GWAS results and observed a strong signal in some diseases (schizophrenia and type 2 diabetes) but not others. We also explored the shared genetic component in overlapping phenotypes related to inflammatory bowel disease (Crohn disease [CD] and ulcerative colitis [UC]) and diabetic nephropathy (macroalbuminuria and end-stage renal disease [ESRD]). Although the signal was still present when both CD and UC were jointly analyzed, the signal was lost when macroalbuminuria and ESRD were jointly analyzed, suggesting that these phenotypes should best be studied separately. Thus, our method may also help guide the design of future genetic studies of various traits and diseases.