Exome Sequencing in Suspected Monogenic Dyslipidemias

Abstract BACKGROUND: -Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We utilized this technique in an attempt to identify novel genes underlying monogenic dyslipidemias. METHODS AND RESULTS: -We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein (LDL) cholesterol (after candidate gene sequencing excluded known genetic causes for high LDL cholesterol families) or high-density lipoprotein (HDL) cholesterol. We used standard analytic approaches to identify candidate variants and also assigned a polygenic score to each individual in order to account for their burden of common genetic variants known to influence lipid levels. In nine families, we identified likely pathogenic variants in known lipid genes (ABCA1, APOB, APOE, LDLR, LIPA, and PCSK9); however, we were unable to identify obvious genetic etiologies in the remaining 32 families despite follow-up analyses. We identified three factors that limited novel gene discovery: (1) imperfect sequencing coverage across the exome hid potentially causal variants; (2) large numbers of shared rare alleles within families obfuscated causal variant identification; and (3) individuals from 15% of families carried a significant burden of common lipid-related alleles, suggesting complex inheritance can masquerade as monogenic disease. CONCLUSIONS: -We identified the genetic basis of disease in nine of 41 families; however, none of these represented novel gene discoveries. Our results highlight the promise and limitations of exome sequencing as a discovery technique in suspected monogenic dyslipidemias. Considering the confounders identified may inform the design of future exome sequencing studies

Genetic predisposition may not improve prediction of cardiac surgery-associated acute kidney injury

Background: The recent integration of genomic data with electronic health records has enabled large scale genomic studies on a variety of perioperative complications, yet genome-wide association studies on acute kidney injury have been limited in size or confounded by composite outcomes. Genome-wide association studies can be leveraged to create a polygenic risk score which can then be integrated with traditional clinical risk factors to better predict postoperative complications, like acute kidney injury.Methods: Using integrated genetic data from two academic biorepositories, we conduct a genome-wide association study on cardiac surgery-associated acute kidney injury. Next, we develop a polygenic risk score and test the predictive utility within regressions controlling for age, gender, principal components, preoperative serum creatinine, and a range of patient, clinical, and procedural risk factors. Finally, we estimate additive variant heritability using genetic mixed models.Results: Among 1,014 qualifying procedures at Vanderbilt University Medical Center and 478 at Michigan Medicine, 348 (34.3%) and 121 (25.3%) developed AKI, respectively. No variants exceeded genome-wide significance (p < 5 × 10−8) threshold, however, six previously unreported variants exceeded the suggestive threshold (p < 1 × 10−6). Notable variants detected include: 1) rs74637005, located in the exonic region of NFU1 and 2) rs17438465, located between EVX1 and HIBADH. We failed to replicate variants from prior unbiased studies of post-surgical acute kidney injury. Polygenic risk was not significantly associated with post-surgical acute kidney injury in any of the models, however, case duration (aOR = 1.002, 95% CI 1.000–1.003, p = 0.013), diabetes mellitus (aOR = 2.025, 95% CI 1.320–3.103, p = 0.001), and valvular disease (aOR = 0.558, 95% CI 0.372–0.835, p = 0.005) were significant in the full model.Conclusion: Polygenic risk score was not significantly associated with cardiac surgery-associated acute kidney injury and acute kidney injury may have a low heritability in this population. These results suggest that susceptibility is only minimally influenced by baseline genetic predisposition and that clinical risk factors, some of which are modifiable, may play a more influential role in predicting this complication. The overall impact of genetics in overall risk for cardiac surgery-associated acute kidney injury may be small compared to clinical risk factors

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant)

LabWAS: Novel findings and study design recommendations from a meta-analysis of clinical labs in two independent biobanks.

Phenotypes extracted from Electronic Health Records (EHRs) are increasingly prevalent in genetic studies. EHRs contain hundreds of distinct clinical laboratory test results, providing a trove of health data beyond diagnoses. Such lab data is complex and lacks a ubiquitous coding scheme, making it more challenging than diagnosis data. Here we describe the first large-scale cross-health system genome-wide association study (GWAS) of EHR-based quantitative laboratory-derived phenotypes. We meta-analyzed 70 lab traits matched between the BioVU cohort from the Vanderbilt University Health System and the Michigan Genomics Initiative (MGI) cohort from Michigan Medicine. We show high replication of known association for these traits, validating EHR-based measurements as high-quality phenotypes for genetic analysis. Notably, our analysis provides the first replication for 699 previous GWAS associations across 46 different traits. We discovered 31 novel associations at genome-wide significance for 22 distinct traits, including the first reported associations for two lab-based traits. We replicated 22 of these novel associations in an independent tranche of BioVU samples. The summary statistics for all association tests are freely available to benefit other researchers. Finally, we performed mirrored analyses in BioVU and MGI to assess competing analytic practices for EHR lab traits. We find that using the mean of all available lab measurements provides a robust summary value, but alternate summarizations can improve power in certain circumstances. This study provides a proof-of-principle for cross health system GWAS and is a framework for future studies of quantitative EHR lab traits

DataSheet1_Genetic predisposition may not improve prediction of cardiac surgery-associated acute kidney injury.pdf

Background: The recent integration of genomic data with electronic health records has enabled large scale genomic studies on a variety of perioperative complications, yet genome-wide association studies on acute kidney injury have been limited in size or confounded by composite outcomes. Genome-wide association studies can be leveraged to create a polygenic risk score which can then be integrated with traditional clinical risk factors to better predict postoperative complications, like acute kidney injury.Methods: Using integrated genetic data from two academic biorepositories, we conduct a genome-wide association study on cardiac surgery-associated acute kidney injury. Next, we develop a polygenic risk score and test the predictive utility within regressions controlling for age, gender, principal components, preoperative serum creatinine, and a range of patient, clinical, and procedural risk factors. Finally, we estimate additive variant heritability using genetic mixed models.Results: Among 1,014 qualifying procedures at Vanderbilt University Medical Center and 478 at Michigan Medicine, 348 (34.3%) and 121 (25.3%) developed AKI, respectively. No variants exceeded genome-wide significance (p −8) threshold, however, six previously unreported variants exceeded the suggestive threshold (p −6). Notable variants detected include: 1) rs74637005, located in the exonic region of NFU1 and 2) rs17438465, located between EVX1 and HIBADH. We failed to replicate variants from prior unbiased studies of post-surgical acute kidney injury. Polygenic risk was not significantly associated with post-surgical acute kidney injury in any of the models, however, case duration (aOR = 1.002, 95% CI 1.000–1.003, p = 0.013), diabetes mellitus (aOR = 2.025, 95% CI 1.320–3.103, p = 0.001), and valvular disease (aOR = 0.558, 95% CI 0.372–0.835, p = 0.005) were significant in the full model.Conclusion: Polygenic risk score was not significantly associated with cardiac surgery-associated acute kidney injury and acute kidney injury may have a low heritability in this population. These results suggest that susceptibility is only minimally influenced by baseline genetic predisposition and that clinical risk factors, some of which are modifiable, may play a more influential role in predicting this complication. The overall impact of genetics in overall risk for cardiac surgery-associated acute kidney injury may be small compared to clinical risk factors.</p

Sex-specific and pleiotropic effects underlying kidney function identified from GWAS meta-analysis

Chronic kidney disease (CKD) is a growing health burden currently affecting 10–15% of adults worldwide. Estimated glomerular filtration rate (eGFR) as a marker of kidney function is commonly used to diagnose CKD. We analyze eGFR data from the Nord-Trøndelag Health Study and Michigan Genomics Initiative and perform a GWAS meta-analysis with public summary statistics, more than doubling the sample size of previous meta-analyses. We identify 147 loci (53 novel) associated with eGFR, including genes involved in transcriptional regulation, kidney development, cellular signaling, metabolism, and solute transport. Additionally, sex-stratified analysis identifies one locus with more significant effects in women than men. Using genetic risk scores constructed from these eGFR meta-analysis results, we show that associated variants are generally predictive of CKD with only modest improvements in detection compared with other known clinical risk factors. Collectively, these results yield additional insight into the genetic factors underlying kidney function and progression to CKD

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease Item Type Article Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

Abstract Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. Zanoni et al. Page 2 P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant). The strong inverse association between amounts of high-density lipoprotein (HDL) cholesterol (HDL-C) and coronary heart disease (CHD) risk has generated interest in a potential causal relationship between HDL metabolism and CHD. However, clinical trials with drugs that raise HDL-C levels, niacin and cholesteryl ester transfer protein (CETP) inhibitors, have produced disappointing results ( 1 ). Furthermore, recent studies of human genetic variants that are associated with HDL-C levels have generally failed to show association with CHD ( 2 , 3 ). Most notably, a loss-of-function variant in LIPG, a gene encoding an endothelial lipase that, in the heterozygous state, raises HDL-C by ~5 mg/dl, was found to have no association with CHD ( 4 ). Although these previous studies suggest that higher HDL-C levels may not be causally protective against CHD, we reasoned that additional human genetic analyses might provide mechanistic insight into the complex relationship between HDL and CHD. The scavenger receptor class BI (SR-BI), encoded by the gene SCARB1, was discovered to be an HDL receptor two decades ago ( 5 ). SR-BI promotes the selective uptake of HDL cholesteryl esters (HDL-CEs) into cells, particularly hepatocytes and steroidogenic cells ( 5 , 6 ). In mice, overexpression of SR-BI in the liver reduces levels of HDL-C ( 7 -10 ), and genetic deletion of SR-BI results in higher HDL-C levels ( 11 -13 ). Remarkably, these genetic manipulations in mice have effects on atherosclerosis opposite to those predicted by human epidemiological data: Overexpression reduces atherosclerosis despite the lower HDL-C levels ( 14 -16 ), and gene deletion increases atherosclerosis despite the higher HDL-C levels ( 17 -20 ). One potential explanation relates to the flux of cholesterol from macrophages through the reverse cholesterol transport (RCT) pathway; SR-BI overexpression increases macrophage RCT, and SR-BI knockout reduces macrophage RCT ( 21 ). The human relevance of these observations has been unclear. Identification of SCARB1 P376L homozygote and association with extremely high HDL-C We hypothesized that humans with extremely high levels of HDL-C may harbor loss-offunction variants in SCARB1 and undertook a targeted resequencing discovery experiment in 328 participants with very high HDL-C (&gt;95th percentile, mean HDL-C of 106.8 mg/dl) and a control group of 398 subjects with low HDL-C (&lt;25th percentile, mean HDL-C of 30.4 mg/dl). In this cohort, we sequenced the exons of ~990 genes located within 300 kb of each of the 95 loci with significant associations (P &lt; 5 × 10 −8 ) with plasma lipid levels identified by the Global Lipids Genetics Consortium as of 2010 C&gt;T, p.P376L, rs74830677), a 67-year-old female with an HDL-C of 152 mg/dl, and confirmed this finding by Sanger sequencing. This subject harbored no mutations in other high HDL-C genes such as CETP and LIPG. In addition to this homozygote, four P376L heterozygotes were identified by targeted sequencing in the high HDL-C group; no heterozygotes were found in the low HDL-C group (P =0.008, Fisher&apos;s exact test). Zanoni et al. Page 3 To identify additional P376L carriers, we genotyped an expanded cohort of very high versus low HDL-C subjects. Among 524 additional subjects with very high HDL-C (mean HDL-C 95.0 mg/dl), we identified 11 heterozygotes for P376L; whereas among 758 subjects with low HDL-C (mean HDL-C 33.5 mg/dl), we identified 3 heterozygotes. In total, our combined sequencing and genotyping for discovery of the P376L variant showed that this variant is significantly overrepresented in subjects with high HDL-C [minor allele frequency (MAF) = 0.010 in high HDL-C versus 0.0013 in low HDL-C controls, P = 0.000127, Fisher&apos;s exact test, Because this variant is present on the exome array, we expanded our analysis to the Global Lipid Genetics Consortium exome array data in &gt;300,000 individuals. The P376L variant was very rare in this population (MAF of ~0.0003). It was significantly associated with higher HDL-C levels with a relatively large effect size (beta = 8.4 mg/dl; P =1.4 × 10 −15 ). Notably, this variant was not associated with plasma levels of low-density lipoprotein cholesterol (LDL-C) or triglycerides (TGs) (table S1). Thus, we conclude that SCARB1 P376L is associated specifically with elevated HDL-C levels. HDL-related phenotypes of SCARB1 P376L homozygote and heterozygotes We next recruited the P376L homozygote, eight heterozygous carriers, and both high HDL-C and normal HDL-C noncarrier controls for deep phenotyping of HDL metabolism and related traits. All of the P376L study participants were of European ancestry, almost exclusively of Ashkenazi Jewish descent. Clinical characteristics and lipid profiles of the subjects are reported in SCARB1 P376L results in complete loss of function of SR-BI Given the profound HDL phenotype of the P376L carriers, we sought to understand the impact of the variant on SR-BI function. We generated induced pluripotent stem cells (iPSCs) using peripheral blood mononuclear cells from the P376L homozygote and a noncarrier control. We next differentiated these cells into hepatocyte-like cells (HLCs) to study HDL metabolism in the setting of endogenous cellular SCARB1 expression. HLCs differentiated through this protocol recapitulate phenotypes of cultured primary hepatocytes Zanoni et al. Page 4 such as albumin and VLDL (very low density lipoprotein) secretion ( 23 -26 ). The cell lines from the control donor and the P376L homozygous subject demonstrated expression of hepatocyte-specific genes ALB (albumin) and AFP (alpha-fetoprotein) and exhibited comparable SCARB1 gene expression ( To evaluate the physiological impact of the P376L variant on HDL-C levels and catabolism in vivo, we used adeno-associated virus (AAV) vectors to direct hepatic overexpression of WT SR-BI or the P376L variant in mice with depleted Scarb1 [Scarb1 knockout (KO) mice]. The two groups of mice demonstrated similar hepatic expression levels of Scarb1 mRNA ( We hypothesized that the markedly reduced HDL-CE uptake could be because of aberrant processing of the P376L SR-BI protein, which leads to impaired cell surface localization. To test this, we isolated cell surface proteins from COS7 cells transfected with WT and P376L SR-BI using biotinylation and found markedly reduced cell surface SR-BI in the P376L transfected cell lysates after streptavidin cell surface protein pull-down assays ( SCARB1 P376L is associated with increased risk of CHD in humans Despite a profound increase in HDL-C, SR-BI deficiency in mice causes accelerated atherosclerosis ( 17 -20 ). The relationship of reduced SR-BI function to atherosclerotic cardiovascular disease in humans has not been established. The P376L homozygous subject did not have clinical CHD, but her carotid intimal-medial thickness (cIMT) was 0.789 mm (left-right average), which is in the &gt;75th percentile for females of her age; in addition, she had detectable plaque throughout the left internal carotid artery and at the bifurcation of her right internal carotid artery. cIMT measurements were not significantly different in the P376L heterozygotes compared with both groups of controls ( To achieve greater statistical power to address this question, we performed a meta-analysis of large exome array genotyping studies of CHD cases and healthy controls to determine the relationship of the P376L variant with risk of CHD ( Discussion Studies of mice have provided important insights into the effects of SR-BI on HDL metabolism, RCT, and atherosclerosis. These studies revealed that overexpression of SR-BI reduces HDL-C ( 7 -10 ) and reduces atherosclerosis ( 14 -16 ), whereas gene deletion of SR-BI increases HDL-C ( 11 -13 ) and accelerates atherosclerosis ( 17 -20 ). The clinical relevance of these findings has remained uncertain, however. Studies of injected labeled HDL-CE in humans suggested that the majority of the HDL-CE was transported to the liver via CETPmediated exchange to apoB-containing lipoproteins rather than by direct uptake from HDL by the liver ( 30 ), which brings into question the importance of hepatic SR-BI in human physiology. Common genetic variants near the SCARB1 locus were found to be significantly associated with plasma HDL-C levels, which suggests that SR-BI may play a role in HDL metabolism in humans ( 22 , 31 ). A family with a rare SCARB1 variant in which serine replaces proline 297 (P297S) was reported in which the heterozygous carriers of the variant had modestly elevated HDL-C levels ( 31 ). However, the variant retains substantial SR-BI activity, no homozygotes were identified, the apparent effect on HDL-C was modest, and there was insufficient power to address its effects on atherosclerosis. Through sequencing of subjects with extremely high plasma levels of HDL-C, we identified a homozygote for a P376L variant in SR-BI. Our complementary approaches consistently Zanoni et al. Page 6 demonstrated that this variant confers virtually complete loss of function of SR-BI. Our results demonstrate many similarities in the consequences of SR-BI deficiency on HDL composition between mice and humans, including a shift toward large, buoyant HDL particles and a significant increase in apoA-I, but not apoA-II, in plasma and HDL ( 12 , 32 , 33 ). The homozygote is a woman who had two healthy children without fertility issues or delivery complications, which suggests that, in humans, SR-BI deficiency may not impair reproductive function in the same manner as it does in mice ( 18 , 34 ). In mice, SR-BImediated CE uptake from HDL is a critical process underlying steroid hormone synthesis in adrenal and gonadal tissues, and SR-BI deficiency alters adrenal cholesterol content, impairs adrenal glucocorticoid response under stress, and can lead to fasting-induced hypoglycemia ( 6 , 35 , 36 ). We did not observe any differences in fasting glucose, serum cortisol, adrenocorticotropic hormone, or female gonadal hormones in P376L heterozygous subjects versus controls, and we saw only a modest increase in testosterone in male P376L heterozygotes relative to noncarriers. We postulate that differences in expression or capacity for up-regulation of apoB-containing lipoprotein receptors relative to SR-BI between mouse models and humans in steroidogenic tissues may account, at least partially, for the lack of recapitulation of some of the phenotypes of SR-BI deficiency in mice. We also observed no differences in platelet levels, cholesterol content, and activation from the P376L carriers, despite reports of thrombocytopenia and altered platelet activity in Scarb1 KO mice ( 31 ). These results suggest a relatively different contribution of SR-BI to platelet function between mice and humans. Note that the phenotypes of human SCARB1 P376L homozygote (elevated HDL-C and large HDL particles but relatively normal steroidogenesis, reproductive viability, and platelet function) are comparable to those observed in mice lacking PDZ domain containing 1 (PDZK1), an adaptor protein for SR-BI ( 37 ). Perhaps the most important finding of our study is that, despite the elevation in HDL-C, P376L carriers exhibit increased risk of CHD, as do Scarb1 KO mice. Our results are consistent with a growing theme in HDL biology that steady-state more important than absolute levels. Using an in vivo assay of macrophage RCT, we previously showed that Scarb1 KO mice have impaired macrophage RCT even though they have elevated HDL-C levels ( 21 ). Our results suggest that reduced hepatic SR-BI function in humans causes impaired RCT, which leads to increased risk of CHD despite elevation in HDL-C levels. However, SR-BI is also expressed in vascular cell types, including endothelial cells, vascular smooth muscle cells, and macrophages, where it could have protective effects against atherosclerosis as well ( 38 , 39 ). Our results are also consistent with the previously suggested concept ( 39 ) that up-regulation or enhancement of SR-BI could be a novel therapeutic approach to reducing CHD risk in the general population