👤 Brent J Small

Fatty acids are important as structural components, energy sources, and signaling mediators. While studies have extensively explored genetic regulation of fatty acids in serum and other bodily fluids, their regulation within adipose tissue, a crucial regulator of cardiovascular and metabolic health, remains unclear. Here, we investigated the genetic regulation of 18 fatty acids in subcutaneous adipose tissue from 569 female twins from TwinsUK. Using twin models, the heritability of fatty acids ranged from 5% to 59%, indicating a substantial genetic regulation of fatty acid levels within adipose tissue, which was also tissue specific in many cases. Genome-wide association studies identified 10 significant loci, in SCD, ADAMTSL1, ZBTB41, SNTB1, EXOC6B, ACSL3, LINC02055, MKRN2/TSEN2, FADS1, and HAPLN across 13 fatty acids or fatty acid product-to-precursor ratios. Using adipose gene expression and methylation, which were concurrently measured in these samples, we detected five fatty acid-associated signals that colocalized with expression quantitative trait locus (eQTL) and methylation quantitative trait locus (meQTL) signals, highlighting fatty acids that are regulated by molecular processes within adipose tissue. We explored links between polygenic scores of common metabolic traits and adipose fatty acid levels and identified associations between polygenic scores of BMI, body-fat distribution, and triglycerides and several fatty acids, indicating these risk scores impact local adipose tissue content. Overall, our results identified local genetic regulation of fatty acids within adipose tissue and highlighted their links with renal and cardio-metabolic health. Show less

Base editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.). Show less

Multivalent pneumococcal vaccines have been developed successfully to combat invasive pneumococcal diseases (IPD) and reduce the associated healthcare burden. These vaccines employ pneumococcal capsular polysaccharides (PnPs), either conjugated or unconjugated, as antigens to provide serotype-specific protection. Pneumococcal capsular polysaccharides used for vaccine often contain residual levels of cell wall polysaccharides (C-Ps), which can generate a non-serotype specific immune response and complicate the desired serotype-specific immunity. Therefore, the C-P level in a pneumococcal vaccine needs to be controlled in the vaccine process and the anti C-P responses need to be dialed out in clinical assays. Currently, two types of cell-wall polysaccharide structures have been identified: a mono-phosphocholine substituted cell-wall polysaccharide C-Ps1 and a di-phosphocholine substituted C-Ps2 structure. In our effort to develop a next-generation novel pneumococcal conjugate vaccine (PCV), we have generated a monoclonal antibody (mAb) specific to cell-wall polysaccharide C-Ps2 structure. An antibody-enhanced HPLC assay (AE-HPLC) has been established for serotype-specific quantification of pneumococcal polysaccharides in our lab. With the new anti C-Ps2 mAb, we herein extend the AE-HPLC assay to the quantification and identification of C-Ps2 species in pneumococcal polysaccharides used for vaccines. Show less

Aortic stenosis (AS) has no approved medical treatment. Identifying etiological pathways for AS could identify pharmacological targets. To identify novel genetic loci and pathways associated with AS. This genome-wide association study used a case-control design to evaluate 44 703 participants (3469 cases of AS) of self-reported European ancestry from the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (from January 1, 1996, to December 31, 2015). Replication was performed in 7 other cohorts totaling 256 926 participants (5926 cases of AS), with additional analyses performed in 6942 participants from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Follow-up biomarker analyses with aortic valve calcium (AVC) were also performed. Data were analyzed from May 1, 2017, to December 5, 2019. Genetic variants (615 643 variants) and polyunsaturated fatty acids (ω-6 and ω-3) measured in blood samples. Aortic stenosis and aortic valve replacement defined by electronic health records, surgical records, or echocardiography and the presence of AVC measured by computed tomography. The mean (SD) age of the 44 703 GERA participants was 69.7 (8.4) years, and 22 019 (49.3%) were men. The rs174547 variant at the FADS1/2 locus was associated with AS (odds ratio [OR] per C allele, 0.88; 95% CI, 0.83-0.93; P = 3.0 × 10-6), with genome-wide significance after meta-analysis with 7 replication cohorts totaling 312 118 individuals (9395 cases of AS) (OR, 0.91; 95% CI, 0.88-0.94; P = 2.5 × 10-8). A consistent association with AVC was also observed (OR, 0.91; 95% CI, 0.83-0.99; P = .03). A higher ratio of arachidonic acid to linoleic acid was associated with AVC (OR per SD of the natural logarithm, 1.19; 95% CI, 1.09-1.30; P = 6.6 × 10-5). In mendelian randomization, increased FADS1 liver expression and arachidonic acid were associated with AS (OR per unit of normalized expression, 1.31 [95% CI, 1.17-1.48; P = 7.4 × 10-6]; OR per 5-percentage point increase in arachidonic acid for AVC, 1.23 [95% CI, 1.01-1.49; P = .04]; OR per 5-percentage point increase in arachidonic acid for AS, 1.08 [95% CI, 1.04-1.13; P = 4.1 × 10-4]). Variation at the FADS1/2 locus was associated with AS and AVC. Findings from biomarker measurements and mendelian randomization appear to link ω-6 fatty acid biosynthesis to AS, which may represent a therapeutic target. Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

The Million Veteran Program (MVP) was established in 2011 as a national research initiative to determine how genetic variation influences the health of US military veterans. Here we genotyped 312,571 MVP participants using a custom biobank array and linked the genetic data to laboratory and clinical phenotypes extracted from electronic health records covering a median of 10.0 years of follow-up. Among 297,626 veterans with at least one blood lipid measurement, including 57,332 black and 24,743 Hispanic participants, we tested up to around 32 million variants for association with lipid levels and identified 118 novel genome-wide significant loci after meta-analysis with data from the Global Lipids Genetics Consortium (total n > 600,000). Through a focus on mutations predicted to result in a loss of gene function and a phenome-wide association study, we propose novel indications for pharmaceutical inhibitors targeting PCSK9 (abdominal aortic aneurysm), ANGPTL4 (type 2 diabetes) and PDE3B (triglycerides and coronary disease). Show less

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 Show less

In a whole-exome sequencing study of multiplex Alzheimer's disease (AD) families, we investigated three neuronal ceroid lipofuscinosis genes that have been linked to retromer, an intracellular trafficking pathway associated with AD: ceroid lipofuscinosis 3 (CLN3), ceroid lipofuscinosis 5 (CLN5), and cathepsin D (CTSD). We identified a missense variant in CLN5 c.A959G (p.Asn320Ser) that segregated with AD. We find that this variant causes glycosylation defects in the expressed protein, which causes it to be retained in the endoplasmic reticulum with reduced delivery to the endolysosomal compartment, CLN5's normal cellular location. The AD-associated CLN5 variant is shown here to reduce the normal processing of cathepsin D and to decrease levels of full-length amyloid precursor protein (APP), suggestive of a defect in retromer-dependent trafficking. Show less

Increased adiposity is a hallmark of obesity and overweight, which affect 2.2 billion people world-wide. Understanding the genetic and molecular mechanisms that underlie obesity-related phenotypes can help to improve treatment options and drug development. Here we perform promoter Capture Hi-C in human adipocytes to investigate interactions between gene promoters and distal elements as a transcription-regulating mechanism contributing to these phenotypes. We find that promoter-interacting elements in human adipocytes are enriched for adipose-related transcription factor motifs, such as PPARG and CEBPB, and contribute to heritability of cis-regulated gene expression. We further intersect these data with published genome-wide association studies for BMI and BMI-related metabolic traits to identify the genes that are under genetic cis regulation in human adipocytes via chromosomal interactions. This integrative genomics approach identifies four cis-eQTL-eGene relationships associated with BMI or obesity-related traits, including rs4776984 and MAP2K5, which we further confirm by EMSA, and highlights 38 additional candidate genes. Show less

We screened variants on an exome-focused genotyping array in >300,000 participants (replication in >280,000 participants) and identified 444 independent variants in 250 loci significantly associated with total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), and/or triglycerides (TG). At two loci (JAK2 and A1CF), experimental analysis in mice showed lipid changes consistent with the human data. We also found that: (i) beta-thalassemia trait carriers displayed lower TC and were protected from coronary artery disease (CAD); (ii) excluding the CETP locus, there was not a predictable relationship between plasma HDL-C and risk for age-related macular degeneration; (iii) only some mechanisms of lowering LDL-C appeared to increase risk for type 2 diabetes (T2D); and (iv) TG-lowering alleles involved in hepatic production of TG-rich lipoproteins (TM6SF2 and PNPLA3) tracked with higher liver fat, higher risk for T2D, and lower risk for CAD, whereas TG-lowering alleles involved in peripheral lipolysis (LPL and ANGPTL4) had no effect on liver fat but decreased risks for both T2D and CAD. Show less

Emerging technologies based on mass spectrometry or nuclear magnetic resonance enable the monitoring of hundreds of small metabolites from tissues or body fluids. Profiling of metabolites can help elucidate causal pathways linking established genetic variants to known disease risk factors such as blood lipid traits. We applied statistical methodology to dissect causal relationships between single nucleotide polymorphisms, metabolite concentrations, and serum lipid traits, focusing on 95 genetic loci reproducibly associated with the four main serum lipids (total-, low-density lipoprotein-, and high-density lipoprotein- cholesterol and triglycerides). The dataset used included 2,973 individuals from two independent population-based cohorts with data for 151 small molecule metabolites and four main serum lipids. Three statistical approaches, namely conditional analysis, Mendelian randomization, and structural equation modeling, were compared to investigate causal relationship at sets of a single nucleotide polymorphism, a metabolite, and a lipid trait associated with one another. A subset of three lipid-associated loci (FADS1, GCKR, and LPA) have a statistically significant association with at least one main lipid and one metabolite concentration in our data, defining a total of 38 cross-associated sets of a single nucleotide polymorphism, a metabolite and a lipid trait. Structural equation modeling provided sufficient discrimination to indicate that the association of a single nucleotide polymorphism with a lipid trait was mediated through a metabolite at 15 of the 38 sets, and involving variants at the FADS1 and GCKR loci. These data provide a framework for evaluating the causal role of components of the metabolome (or other intermediate factors) in mediating the association between established genetic variants and diseases or traits. Show less

Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h(2)median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner. Show less

The basic helix-loop-helix transcriptional repressor Hairy-related transcription factor 2 (Hrt2) is expressed in ventricular, but not atrial, cardiomyocytes, and in endothelial and vascular smooth muscle cells. Mice homozygous for a null mutation of Hrt2 die perinatally from a spectrum of cardiac abnormalities, raising questions about the specific functions of this transcriptional regulator in individual cardiac cell lineages. Using a conditional Hrt2 null allele, we show that cardiomyocyte-specific deletion of Hrt2 in mice results in ectopic activation of atrial genes in ventricular myocardium with an associated impairment of cardiac contractility and a unique distortion in morphology of the right ventricular chamber. Consistent with the atrialization of ventricular gene expression in Hrt2 mutant mice, forced expression of Hrt2 in atrial cardiomyocytes is sufficient to repress atrial cardiac genes. These findings reveal a ventricular myocardial cell-autonomous function for Hrt2 in the suppression of atrial cell identity and the maintenance of postnatal cardiac function. Show less

Amphipathic alpha-helices are the main structure and the major lipid binding motif of exchangeable apolipoproteins. To understand how these apolipoproteins behave at an hydrophobic lipoprotein interface, the interfacial properties of a consensus sequence peptide (CSP) derived from three exchangeable apolipoproteins (A-I, A-IV, and E) were studied using an oil drop tensiometer at air/water (A/W) and dodecane/water (DD/W) interfaces. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-amino acid tandem repeat sequences that form amphipathic alpha-helices. CSP, when added into the aqueous phase, lowered the interfacial tension (gamma) of A/W and DD/W in a concentration-dependent fashion. The gammaA/W was lowered approximately 24 mn/m, and gammaDD/W approximately 31 mn/m, indicating a greater affinity of CSP for DD/W. Using the Gibbs equation for surface, the surface area per CSP molecule was estimated at approximately 702 A2 ( approximately 16 A2/amino acid) on A/W and approximately 622 A2 on DD/W ( approximately 14 A2/amino acid) suggesting that adsorbed CSP lies flat with alpha-helices in the plane of both interfaces. At equilibrium gamma, CSP desorbed from the interface when compressed and re-adsorbed when expanded. The adsorption rate was concentration-dependent, but the desorption rate was not. Less CSP desorbed from DD/W than A/W indicating that CSP has higher affinity for DD/W. Dynamic analysis of elasticity shows that the faster the oscillation period (4, 8 s) and the lower the oscillation amplitude the more elastic the surfaces. CSP can be compressed 6-12% while remaining on the surface, but large increases in pressure eject it from the surface. We suggest that surface pressure-mediated desorption and readsorption of amphipathic alpha-helices provide lipoprotein stability during remodeling reactions in plasma. Show less