Analysis of Protein Structure-Function in Vivo: ADENOVIRUS-MEDIATED TRANSFER OF LIPASE LID MUTANTS IN HEPATIC LIPASE-DEFICIENT MICE

Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes involved in the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Despite their similarities, the role that each of these two lipases play in the metabolism of triglyceride-rich lipoproteins and high density lipoproteins is distinct. In order to identify structural domains that may confer the different substrate specificities between HL and LPL, we have utilized a novel approach for performing structure-function analysis of a protein, in vivo, by using recombinant adenovirus vectors to express native and mutant enzymes in an animal model for a human genetic deficiency. HL-deficient mice (n = 19) characterized by increased plasma cholesterol and phospholipid concentrations were injected with adenovirus expressing luciferase (rLucif-AdV), native hepatic (rHL-AdV), and lipoprotein lipase (rLPL-AdV) or lipase mutants in which the lid covering the catalytic site of either enzyme was exchanged (rHL+LPL lid-AdV and rLPL+HL lid-AdV). Mice injected with rLucif-AdV had no changes in post-heparin HL and LPL activities (217 +/- 29 and 7 +/- 2 nmol/min/ml, respectively) as well as plasma lipids. Despite expression of similar levels of post-heparin plasma lipase activity on day 5 post-adenovirus infusion (9806 +/- 915 and 9677 +/- 2033 nmol/min/ml, respectively) mice injected with rHL-AdV or rHL+LPL lid-AdV demonstrated marked differences in the reduction of plasma phospholipids (70% and 32%, respectively, p < 0.005). Similarly, despite post-heparin plasma lipolytic activities of 4495 +/- 534 and 4844 +/- 1336 nmol/min/ml, injection of rLPL-AdV or rLPL+HL lid-AdV resulted in phospholipid reductions of 31% and 81% (p < 0.005). Exchange of the lipase lid did not significantly alter plasma triglyceride concentrations. Thus, preferential in vivo hydrolysis of phospholipids was demonstrated in animals expressing lipases containing the HL lid but not the LPL lid. These studies identify the lipase lid as a major structural motif responsible for conferring the different in vivo phospholipase activities between HL and LPL, a function which may modulate the distinct physiological roles of these two similar lipolytic enzymes in lipoprotein metabolism. The use of recombinant adenovirus to express mutant proteins in animal models for human genetic deficiencies represents a powerful, new approach for performing structure-function analysis of proteins in vivo

Whole Genome Sequence Analysis of the Plasma Proteome in Black Adults Provides Novel Insights Into Cardiovascular Disease

BackgroundPlasma proteins are critical mediators of cardiovascular processes and are the targets of many drugs. Previous efforts to characterize the genetic architecture of the plasma proteome have been limited by a focus on individuals of European descent and leveraged genotyping arrays and imputation. Here we describe whole genome sequence analysis of the plasma proteome in individuals with greater African ancestry, increasing our power to identify novel genetic determinants.MethodsProteomic profiling of 1301 proteins was performed in 1852 Black adults from the Jackson Heart Study using aptamer-based proteomics (SomaScan). Whole genome sequencing association analysis was ascertained for all variants with minor allele count ≥5. Results were validated using an alternative, antibody-based, proteomic platform (Olink) as well as replicated in the Multi-Ethnic Study of Atherosclerosis and the HERITAGE Family Study (Health, Risk Factors, Exercise Training and Genetics).ResultsWe identify 569 genetic associations between 479 proteins and 438 unique genetic regions at a Bonferroni-adjusted significance level of 3.8×10-11. These associations include 114 novel locus-protein relationships and an additional 217 novel sentinel variant-protein relationships. Novel cardiovascular findings include new protein associations at the APOE gene locus including ZAP70 (sentinel single nucleotide polymorphism [SNP] rs7412-T, β=0.61±0.05, P=3.27×10-30) and MMP-3 (β=-0.60±0.05, P=1.67×10-32), as well as a completely novel pleiotropic locus at the HPX gene, associated with 9 proteins. Further, the associations suggest new mechanisms of genetically mediated cardiovascular disease linked to African ancestry; we identify a novel association between variants linked to APOL1-associated chronic kidney and heart disease and the protein CKAP2 (rs73885319-G, β=0.34±0.04, P=1.34×10-17) as well as an association between ATTR amyloidosis and RBP4 levels in community-dwelling individuals without heart failure.ConclusionsTaken together, these results provide evidence for the functional importance of variants in non-European populations, and suggest new biological mechanisms for ancestry-specific determinants of lipids, coagulation, and myocardial function

Dynamic incorporation of multiple in silico functional annotations empowers rare variant association analysis of large whole-genome sequencing studies at scale

Large-scale whole-genome sequencing studies have enabled the analysis of rare variants (RVs) associated with complex phenotypes. Commonly used RV association tests have limited scope to leverage variant functions. We propose STAAR (variant-set test for association using annotation information), a scalable and powerful RV association test method that effectively incorporates both variant categories and multiple complementary annotations using a dynamic weighting scheme. For the latter, we introduce ‘annotation principal components’, multidimensional summaries of in silico variant annotations. STAAR accounts for population structure and relatedness and is scalable for analyzing very large cohort and biobank whole-genome sequencing studies of continuous and dichotomous traits. We applied STAAR to identify RVs associated with four lipid traits in 12,316 discovery and 17,822 replication samples from the Trans-Omics for Precision Medicine Program. We discovered and replicated new RV associations, including disruptive missense RVs of NPC1L1 and an intergenic region near APOC1P1 associated with low-density lipoprotein cholesterol

Whole Genome Sequencing Identifies CRISPLD2 as a Lung Function Gene in Children With Asthma

BackgroundAsthma is a common respiratory disorder with a highly heterogeneous nature that remains poorly understood. The objective was to use whole genome sequencing (WGS) data to identify regions of common genetic variation contributing to lung function in individuals with a diagnosis of asthma.MethodsWGS data were generated for 1,053 individuals from trios and extended pedigrees participating in the family-based Genetic Epidemiology of Asthma in Costa Rica study. Asthma affection status was defined through a physician's diagnosis of asthma, and most participants with asthma also had airway hyperresponsiveness (AHR) to methacholine. Family-based association tests for single variants were performed to assess the associations with lung function phenotypes.ResultsA genome-wide significant association was identified between baseline FEV1/FVC ratio and a single-nucleotide polymorphism in the top hit cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) (rs12051168; P&nbsp;= 3.6&nbsp;× 10-8 in the unadjusted model) that retained suggestive significance in the covariate-adjusted model (P&nbsp;= 5.6&nbsp;× 10-6). Rs12051168 was also nominally associated with other related phenotypes: baseline FEV1 (P&nbsp;= 3.3&nbsp;× 10-3), postbronchodilator (PB) FEV1 (7.3&nbsp;× 10-3), and PB FEV1/FVC ratio (P&nbsp;= 2.7&nbsp;× 10-3). The&nbsp;identified baseline FEV1/FVC ratio and rs12051168 association was meta-analyzed and&nbsp;replicated in three independent cohorts in which most participants with asthma also had confirmed AHR (combined weighted z-score P&nbsp;= .015) but not in cohorts without information about AHR.ConclusionsThese findings suggest that using specific asthma characteristics, such as AHR, can help identify more genetically homogeneous asthma subgroups with genotype-phenotype associations that may not be observed in all children with asthma. CRISPLD2 also may be important for baseline lung function in individuals with asthma who also may have AHR

Chromosome Xq23 is associated with lower atherogenic lipid concentrations and favorable cardiometabolic indices

Abstract Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10−72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10−4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10−5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids