Aris Baras

Inactivating Variants in ANGPTL4 and Risk of Coronary Artery Disease

BACKGROUND Higher-than-normal levels of circulating triglycerides are a risk factor for ischemic cardiovascular disease. Activation of lipoprotein lipase, an enzyme that is inhibited by angiopoietin-like 4 (ANGPTL4), has been shown to reduce levels of circulating triglycerides. METHODS We sequenced the exons of ANGPTL4 in samples obtain from 42,930 participants of predominantly European ancestry in the DiscovEHR human genetics study. We performed tests of association between lipid levels and the missense E40K variant (which has been associated with reduced plasma triglyceride levels) and other inactivating mutations. We then tested for associations between coronary artery disease and the E40K variant and other inactivating mutations in 10,552 participants with coronary artery disease and 29,223 controls. We also tested the effect of a human monoclonal antibody against ANGPTL4 on lipid levels in mice and monkeys. RESULTS We identified 1661 heterozygotes and 17 homozygotes for the E40K variant and 75 participants who had 13 other monoallelic inactivating mutations in ANGPTL4. The levels of triglycerides were 13% lower and the levels of high-density lipoprotein (HDL) cholesterol were 7% higher among carriers of the E40K variant than among noncarriers. Carriers of the E40K variant were also significantly less likely than noncarriers to have coronary artery disease (odds ratio, 0.81; 95% confidence interval, 0.70 to 0.92; P=0.002). K40 homozygotes had markedly lower levels of triglycerides and higher levels of HDL cholesterol than did heterozygotes. Carriers of other inactivating mutations also had lower triglyceride levels and higher HDL cholesterol levels and were less likely to have coronary artery disease than were noncarriers. Monoclonal antibody inhibition of Angptl4 in mice and monkeys reduced triglyceride levels. CONCLUSIONS Carriers of E40K and other inactivating mutations in ANGPTL4 had lower levels of triglycerides and a lower risk of coronary artery disease than did noncarriers. The inhibition of Angptl4 in mice and monkeys also resulted in corresponding reductions in these values. (Funded by Regeneron Pharmaceuticals.).

Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease

BACKGROUND Loss‐of‐function variants in the angiopoietin‐like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low‐density lipoprotein (LDL) cholesterol, and high‐density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease. METHODS We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss‐of‐function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow‐up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol. RESULTS In the DiscovEHR study, participants with heterozygous loss‐of‐function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss‐of‐function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow‐up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose‐dependent placebo‐adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%. CONCLUSIONS Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878.)

Distribution and clinical impact of functional variants in 50,726 whole-exome sequences from the DiscovEHR study

Unleashing the power of precision medicine Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey et al. found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn et al. extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment. Science, this issue p. 10.1126/science.aaf6814, p. 10.1126/science.aaf7000 More than 50,000 exomes, coupled with electronic health records, inform on medically relevant genetic variants. INTRODUCTION Large-scale genetic studies of integrated health care populations, with phenotypic data captured natively in the documentation of clinical care, have the potential to unveil genetic associations that point the way to new biology and therapeutic targets. This setting also represents an ideal test bed for the implementation of genomics in routine clinical care in service of precision medicine. RATIONALE The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System aims to catalyze genomic discovery and precision medicine by coupling high-throughput exome sequencing to longitudinal electronic health records (EHRs) of participants in Geisinger’s MyCode Community Health Initiative. Here, we describe initial insights from whole-exome sequencing of 50,726 adult participants of predominantly European ancestry using clinical phenotypes derived from EHRs. RESULTS The median duration of EHR data associated with sequenced participants was 14 years, with a median of 87 clinical encounters, 687 laboratory tests, and seven procedures per participant. Forty-eight percent of sequenced individuals had one or more first- or second-degree relatives in the sample, and genome-wide autozygosity was similar to other outbred European populations. We found ~4.2 million single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in loss of gene function (LoF). The overwhelming majority of these genetic variants occurred at a minor allele frequency of ≤1%, and more than half were singletons. Each participant harbored a median of 21 rare predicted LoFs. At this sample size, ~92% of sequenced genes, including genes that encode existing drug targets or confer risk for highly penetrant genetic diseases, harbor rare heterozygous predicted LoF variants. About 7% of sequenced genes contained rare homozygous predicted LoF variants in at least one individual. Linking these data to EHR-derived laboratory phenotypes revealed consequences of partial or complete LoF in humans. Among these were previously unidentified associations between predicted LoFs in CSF2RB and basophil and eosinophil counts, and EGLN1-associated erythrocytosis segregating in genetically identified family networks. Using predicted LoFs as a model for drug target antagonism, we found associations supporting the majority of therapeutic targets for lipid lowering. To highlight the opportunity for genotype-phenotype association discovery, we performed exome-wide association analyses of EHR-derived lipid values, newly implicating rare predicted LoFs, and deleterious missense variants in G6PC in association with triglyceride levels. In a survey of 76 clinically actionable disease-associated genes, we estimated that 3.5% of individuals harbor pathogenic or likely pathogenic variants that meet criteria for clinical action. Review of the EHR uncovered findings associated with the monogenic condition in ~65% of pathogenic variant carriers’ medical records. CONCLUSION The findings reported here demonstrate the value of large-scale sequencing in an integrated health system population, add to the knowledge base regarding the phenotypic consequences of human genetic variation, and illustrate the challenges and promise of genomic medicine implementation. DiscovEHR provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic target discovery. Therapeutic target validation and genomic medicine in DiscovEHR. (A) Associations between predicted LoF variants in lipid drug target genes and lipid levels. Boxes correspond to effect size, given as the absolute value of effect, in SD units; whiskers denote 95% confidence intervals for effect. The size of the box is proportional to the logarithm (base 10) of predicted LoF carriers. (B and C) Prevalence and expressivity of clinically actionable genetic variants in 76 disease genes, according to EHR data. G76, Geisinger-76. The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.

Genetic identification of familial hypercholesterolemia within a single U.S. health care system

Unleashing the power of precision medicine Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey et al. found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn et al. extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment. Science, this issue p. 10.1126/science.aaf6814, p. 10.1126/science.aaf7000 Genomic screening can prompt the diagnosis of familial hypercholesterolemia patients, the majority of whom are receiving inadequate lipid-lowering therapy. INTRODUCTION Familial hypercholesterolemia (FH) is a public health genomics priority but remains underdiagnosed and undertreated despite widespread cholesterol screening. This represents a missed opportunity to prevent FH-associated cardiovascular morbidity and mortality. Pathogenic variants in three genes (LDLR, APOB, and PCSK9) account for the majority of FH cases. We assessed the prevalence and clinical impact of FH-associated genomic variants in 50,726 individuals from the MyCode Community Health Initiative at Geisinger Health System who underwent exome sequencing as part of the DiscovEHR human genetics collaboration with the Regeneron Genetics Center. RATIONALE Genetic testing for FH is uncommon in clinical practice in the United States, and the prevalence of FH variants in U.S. populations has not been well established. We sought to evaluate FH prevalence in a large integrated U.S. health care system using genomic sequencing and electronic health record (EHR) data. We determined the impact of FH variants on low-density lipoprotein cholesterol (LDL-C) levels and coronary artery disease (CAD) risk. We assessed the likelihood of FH variant carriers achieving a presequencing EHR-based FH diagnosis according to established clinical diagnostic criteria. Finally, we examined the rates of statin medication use and outcomes in FH variant carriers. RESULTS Thirty-five known and predicted pathogenic variants in LDLR, APOB, and PCSK9 were identified in 229 individuals. The estimated FH prevalence was 1:256 in unselected participants and 1:118 in participants ascertained via the cardiac catheterization laboratory. FH variants were found in only 2.5% of individuals with severe hypercholesterolemia (maximum EHR-documented LDL-C ≥ 190 mg/dl) in the cohort, and a maximum LDL-C of ≥190 mg/dl was absent in 45% of FH variant carriers. Overall, FH variant carriers had 69 ± 3 mg/dl greater maximum LDL-C than sequenced noncarriers (P = 1.8 × 10−20) and had significantly increased odds of general and premature CAD [odds ratio (OR), 2.6 (P = 4.3 × 10−11) and 3.7 (P = 5.5 × 10−14), respectively]. The increased odds of general and premature CAD were most pronounced in carriers of LDLR predicted loss-of-function variants [OR, 5.5 (P = 7.7 × 10−13) and 10.3 (P = 9.8 × 10−19), respectively]. Fourteen FH variant carriers were deceased; chart review revealed that none of these individuals had a clinical diagnosis of FH. Before genetic testing, only 15% of FH variant carriers had an ICD-10 (International Classification of Diseases, 10th revision) diagnosis code for pure hypercholesterolemia or had been seen in a lipid clinic, suggesting that few had been previously diagnosed with FH. Retrospectively applying Dutch Lipid Clinic Network diagnostic criteria to EHR data, we found presequencing criteria supporting a probable or definite clinical diagnosis of FH in 24% of FH variant carriers, highlighting the limitations of using existing clinical criteria for EHR-based screening in the absence of genetic testing. Active statin use was identified in 58% and high-intensity statin use in 37% of FH variant carriers. Only 46% of carriers currently on statin therapy had a most recent LDL-C level below 100 mg/dl compared to 77% of noncarriers. CONCLUSION In summary, we show that large-scale genomic screening in patients with longitudinal EHR data has the ability to detect FH, uncover and characterize novel pathogenic variants, determine disease prevalence, and enhance overall knowledge of clinical impact and outcomes. The 1:256 prevalence of FH variants in this predominantly European-American cohort is in line with prevalence estimates from recent work in European cohorts. Our findings highlight the undertreatment of FH variant carriers and demonstrate a potential clinical benefit for large-scale sequencing initiatives in service of precision medicine. Prevalence and clinical impact of FH variants in a large U.S. clinical care cohort. (A) Distribution of 229 heterozygous carriers of an FH variant in the DiscovEHR cohort by FH gene. (B) Prevalence of an FH variant in the DiscovEHR cohort and according to recruitment site

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease

BACKGROUND Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17‐beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10‐12) and AST (P=6.2×10‐10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk‐increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS A loss‐of‐function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.)

CLAMMS: a scalable algorithm for calling common and rare copy number variants from exome sequencing data

Motivation: Several algorithms exist for detecting copy number variants (CNVs) from human exome sequencing read depth, but previous tools have not been well suited for large population studies on the order of tens or hundreds of thousands of exomes. Their limitations include being difficult to integrate into automated variant-calling pipelines and being ill-suited for detecting common variants. To address these issues, we developed a new algorithm—Copy number estimation using Lattice-Aligned Mixture Models (CLAMMS)—which is highly scalable and suitable for detecting CNVs across the whole allele frequency spectrum. Results: In this note, we summarize the methods and intended use-case of CLAMMS, compare it to previous algorithms and briefly describe results of validation experiments. We evaluate the adherence of CNV calls from CLAMMS and four other algorithms to Mendelian inheritance patterns on a pedigree; we compare calls from CLAMMS and other algorithms to calls from SNP genotyping arrays for a set of 3164 samples; and we use TaqMan quantitative polymerase chain reaction to validate CNVs predicted by CLAMMS at 39 loci (95% of rare variants validate; across 19 common variant loci, the mean precision and recall are 99% and 94%, respectively). In the Supplementary Materials (available at the CLAMMS Github repository), we present our methods and validation results in greater detail. Availability and implementation: https://github.com/rgcgithub/clamms (implemented in C). Contact: jeffrey.reid@regeneron.com Supplementary information: Supplementary data are available at Bioinformatics online.

Familial Hypercholesterolemia and Type 2 Diabetes in the Old Order Amish

Alleles associated with lower levels of LDL cholesterol (LDL-C) have recently been associated with an increased risk of type 2 diabetes (T2D), highlighting the complex relationship between LDL-C and diabetes. This observation begs the question of whether LDL-C–raising alleles are associated with a decreased risk of T2D. This issue was recently addressed in a large familial hypercholesterolemia (FH) screening study, which reported a lower prevalence of self-reported diabetes in FH subjects than in age-matched relatives without FH. To extend this observation, we tested the association of FH with diabetes status and glycemia in a large Amish population enriched for the FH-associated APOB R3527Q variant that included 640 APOB R3527Q carriers and 4,683 noncarriers. Each copy of the R3527Q T allele was associated with a 74.9 mg/dL increase in LDL-C. There was little difference in T2D prevalence between subjects with (5.2%) and without (4.5%) the R3527Q allele (P = 0.23), and there was no association between R3527Q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test–derived measures. Our data provide no evidence supporting an association between the APOB R3527Q variant and T2D or glycemia and highlight the asymmetry of the LDL-C–T2D relationship and/or the gene/variant-dependent specificity of the LDL-C–T2D association.

Genetic inactivation of ANGPTL4 improves glucose homeostasis and is associated with reduced risk of diabetes

Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85–0.92, p = 6.3 × 10−10), lower fasting glucose, and greater insulin sensitivity. Predicted loss-of-function variants are associated with lower odds of T2D among 32,015 cases and 84,006 controls (odds ratio 0.71, 95% confidence interval 0.49–0.99, p = 0.041). Functional studies in Angptl4-deficient mice confirm improved insulin sensitivity and glucose homeostasis. In conclusion, genetic inactivation of ANGPTL4 is associated with improved glucose homeostasis and reduced risk of T2D.Genetic variation in ANGPTL4 is associated with lipid traits. Here, the authors find that predicted loss-of-function variants in ANGPTL4 are associated with glucose homeostasis and reduced risk of type 2 diabetes and that Angptl4−/− mice on a high-fat diet show improved insulin sensitivity.

Genomic diagnostics within a medically underserved population: efficacy and implications

PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, “family” WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.

Exome Sequencing in Children With Pulmonary Arterial Hypertension Demonstrates Differences Compared With Adults

Background: Pulmonary arterial hypertension (PAH) is a rare disease characterized by pulmonary arteriole remodeling, elevated arterial pressure and resistance, and subsequent heart failure. Compared with adult-onset disease, pediatric-onset PAH is more heterogeneous and often associated with worse prognosis. Although BMPR2 mutations underlie ≈70% of adult familial PAH (FPAH) cases, the genetic basis of PAH in children is less understood. Methods: We performed genetic analysis of 155 pediatric- and 257 adult-onset PAH patients, including both FPAH and sporadic, idiopathic PAH (IPAH). After screening for 2 common PAH risk genes, mutation-negative FPAH and all IPAH cases were evaluated by exome sequencing. Results: We observed similar frequencies of rare, deleterious BMPR2 mutations in pediatric- and adult-onset patients: ≈55% in FPAH and 10% in IPAH patients in both age groups. However, there was significant enrichment of TBX4 mutations in pediatric- compared with adult-onset patients (IPAH: 10/130 pediatric versus 0/178 adult-onset), and TBX4 carriers had younger mean age-of-onset compared with BMPR2 carriers. Mutations in other known PAH risk genes were infrequent in both age groups. Notably, among pediatric IPAH patients without mutations in known risk genes, exome sequencing revealed a 2-fold enrichment of de novo likely gene-damaging and predicted deleterious missense variants. Conclusions: Mutations in known PAH risk genes accounted for ≈70% to 80% of FPAH in both age groups, 21% of pediatric-onset IPAH, and 11% of adult-onset IPAH. Rare, predicted deleterious variants in TBX4 are enriched in pediatric patients and de novo variants in novel genes may explain ≈19% of pediatric-onset IPAH cases.