Sequencing of sporadic Attention-Deficit Hyperactivity Disorder (ADHD) identifies novel and potentially pathogenic de novo variants and excludes overlap with genes associated with autism spectrum disorder.

Attention-Deficit Hyperactivity Disorder (ADHD) has high heritability; however, studies of common variation account for <5% of ADHD variance. Using data from affected participants without a family history of ADHD, we sought to identify de novo variants that could account for sporadic ADHD. Considering a total of 128 families, two analyses were conducted in parallel: first, in 11 unaffected parent/affected proband trios (or quads with the addition of an unaffected sibling) we completed exome sequencing. Six de novo missense variants at highly conserved bases were identified and validated from four of the 11 families: the brain-expressed genes TBC1D9, DAGLA, QARS, CSMD2, TRPM2, and WDR83. Separately, in 117 unrelated probands with sporadic ADHD, we sequenced a panel of 26 genes implicated in intellectual disability (ID) and autism spectrum disorder (ASD) to evaluate whether variation in ASD/ID-associated genes were also present in participants with ADHD. Only one putative deleterious variant (Gln600STOP) in CHD1L was identified; this was found in a single proband. Notably, no other nonsense, splice, frameshift, or highly conserved missense variants in the 26 gene panel were identified and validated. These data suggest that de novo variant analysis in families with independently adjudicated sporadic ADHD diagnosis can identify novel genes implicated in ADHD pathogenesis. Moreover, that only one of the 128 cases (0.8%, 11 exome, and 117 MIP sequenced participants) had putative deleterious variants within our data in 26 genes related to ID and ASD suggests significant independence in the genetic pathogenesis of ADHD as compared to ASD and ID phenotypes. © 2017 Wiley Periodicals, Inc.

PLTP activity inversely correlates with CAAD: effects of PON1 enzyme activity and genetic variants on PLTP activity.

Recent studies have failed to demonstrate a causal cardioprotective effect of HDL cholesterol levels, shifting focus to the functional aspects of HDL. Phospholipid transfer protein (PLTP) is an HDL-associated protein involved in reverse cholesterol transport. This study sought to determine the genetic and nongenetic predictors of plasma PLTP activity (PLTPa), and separately, to determine whether PLTPa predicted carotid artery disease (CAAD). PLTPa was measured in 1,115 European ancestry participants from a case-control study of CAAD. A multivariate logistic regression model was used to elucidate the relationship between PLTPa and CAAD. Separately, a stepwise linear regression determined the nongenetic clinical and laboratory characteristics that best predicted PLTPa. A final stepwise regression considering both nongenetic and genetic variables identified the combination of covariates that explained maximal PLTPa variance. PLTPa was significantly associated with CAAD (7.90 × 10(-9)), with a 9% decrease in odds of CAAD per 1 unit increase in PLTPa (odds ratio = 0.91). Triglyceride levels (P = 0.0042), diabetes (P = 7.28 × 10(-5)), paraoxonase 1 (PON1) activity (P = 0.019), statin use (P = 0.026), PLTP SNP rs4810479 (P = 6.38 × 10(-7)), and PCIF1 SNP rs181914932 (P = 0.041) were all significantly associated with PLTPa. PLTPa is significantly inversely correlated with CAAD. Furthermore, we report a novel association between PLTPa and PON1 activity, a known predictor of CAAD.

Quantification of HDL particle concentration by calibrated ion mobility analysis.

BACKGROUND: It is critical to develop new metrics to determine whether HDL is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P), the size and concentration of HDL in plasma. However, the 2 methods currently used to determine HDL-P yield concentrations that differ >5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA). METHODS: HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. We used a calibration curve constructed with purified proteins to correct for the ionization efficiency of HDL particles. RESULTS: The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n = 40) and cerebrovascular disease (n = 40) participants, 3 subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4 (2.4) µmol/L. HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in participants with cerebrovascular disease (P = 0.002), and this difference remained significant after adjustment for HDL cholesterol concentrations (P = 0.02). CONCLUSIONS: Calibrated IMA accurately determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting that the method could accurately quantify HDL particle concentration. The estimated stoichiometry of apolipoprotein A-I determined by calibrated IMA was 3-4 per HDL particle, in agreement with current structural models. Furthermore, HDL-P was associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.

Novel gene-by-environment interactions: APOB and NPC1L1 variants affect the relationship between dietary and total plasma cholesterol.

Cardiovascular disease (CVD) is the leading cause of death in developed countries. Plasma cholesterol level is a key risk factor in CVD pathogenesis. Genetic and dietary variation both influence plasma cholesterol; however, little is known about dietary interactions with genetic variants influencing the absorption and transport of dietary cholesterol. We sought to determine whether gut expressed variants predicting plasma cholesterol differentially affected the relationship between dietary and plasma cholesterol levels in 1,128 subjects (772/356 in the discovery/replication cohorts, respectively). Four single nucleotide polymorphisms (SNPs) within three genes (APOB, CETP, and NPC1L1) were significantly associated with plasma cholesterol in the discovery cohort. These were subsequently evaluated for gene-by-environment (GxE) interactions with dietary cholesterol for the prediction of plasma cholesterol, with significant findings tested for replication. Novel GxE interactions were identified and replicated for two variants: rs1042034, an APOB Ser4338Asn missense SNP and rs2072183 (in males only), a synonymous NPC1L1 SNP in linkage disequilibrium with SNPs 5' of NPC1L1. This study identifies the presence of novel GxE and gender interactions implying that differential gut absorption is the basis for the variant associations with plasma cholesterol. These GxE interactions may account for part of the "missing heritability" not accounted for by genetic associations.

Novel common and rare genetic determinants of paraoxonase activity: FTO, SERPINA12, and ITGAL.

HDL-associated paraoxonase 1 (PON1) activity is associated with cardiovascular and other human diseases. As the role of genetic variants outside of the PON gene cluster on PON1 activity is unknown, we sought to identify common and rare variants in such loci. We typed 33,057 variants on the CVD chip in 1,362 subjects to test for their effects on adjusted-PON1 activity. Three novel genes (FTO, ITGAL, and SERPINA12) and the PON gene cluster had SNPs associated with PON1 arylesterase (AREase) activity. These loci were carried forward for rare-variant analysis using Exome chip genotypes in an overlapping subset of 1,051 subjects using sequence kernel association testing. PON1 (P = 2.24 × 10(-4)), PON3 (P = 0.022), FTO (P = 0.019), and SERPINA12 (P = 0.039) had both common and rare variants associated with PON1 AREase. ITGAL variants were associated with PON1 activity when using weighted sequence kernel association testing (SKAT) analysis (P = 2.63 × 10(-3)). When adjusting for the initial common variants, SERPINA12 became marginally significant (P = 0.09), whereas all other findings remained significant (P < 0.05), suggesting independent rare-variant effects. We present novel findings that common and rare variants in FTO, SERPINA12, and ITGAL predict PON1 activity. These results further link PON1 to diabetes and inflammation and may inform the role of HDL in human disease.

Dietary fatty acid intake is associated with paraoxonase 1 activity in a cohort-based analysis of 1,548 subjects.

BACKGROUND: Paraoxonase 1 (PON1) is a cardioprotective, HDL-associated glycoprotein enzyme with broad substrate specificity. Our previous work found associations between dietary cholesterol and vitamin C with PON1 activity. The goal of this study was to determine the effect of specific dietary fatty acid (DFA) intake on PON1 activity. METHODS: 1,548 participants with paraoxonase activity measures completed the Harvard Standardized Food Frequency Questionnaire to determine their daily nutrient intake over the past year. Eight saturated, 3 monounsaturated, and 6 polyunsaturated DFAs were measured by the questionnaire. To reduce the number of observations tested, only specific fatty acids that were not highly correlated (r < 0.8) with other DFAs or that were representative of other DFAs through high correlation within each respective group (saturated, monounsaturated, or polyunsaturated) were retained for analysis. Six specific DFA intakes - myristic acid (14 carbon atoms, no double bonds - 14:0), oleic acid (18:1), gadoleic acid (20:1), α-linolenic acid (18:3), arachidonic acid (20:4), and eicosapentaenoic acid (20:5) - were carried forward to stepwise linear regression, which evaluated the effect of each specific DFA on covariate-adjusted PON1 enzyme activity. RESULTS: Four of the 6 tested DFA intakes - myristic acid (p = 0.038), gadoleic acid (p = 6.68 × 10(-7)), arachidonic acid (p = 0.0007), and eicosapentaenoic acid (p = 0.013) - were independently associated with covariate-adjusted PON1 enzyme activity. Myristic acid, a saturated fat, and gadoleic acid, a monounsaturated fat, were both positively associated with PON1 activity. Both of the tested polyunsaturated fats, arachidonic acid and eicosapentaenoic acid, were negatively associated with PON1 activity. CONCLUSIONS: This study presents the largest cohort-based analysis of the relationship between dietary lipids and PON1 enzyme activity. Further research is necessary to elucidate and understand the specific biological mechanisms, whether direct or regulatory, through which DFAs affect PON1 activity.

Dietary cholesterol increases paraoxonase 1 enzyme activity.

HDL-associated paraoxonase 1 (PON1) activity has been consistently associated with cardiovascular and other diseases. Vitamins C and E intake have previously been positively associated with PON1 in a subset of the Carotid Lesion Epidemiology and Risk (CLEAR) cohort. The goal of this study was to replicate these findings and determine whether other nutrient intake affected PON1 activity. To predict nutrient and mineral intake values, 1,402 subjects completed a standardized food frequency survey of their dietary habits over the past year. Stepwise regression was used to evaluate dietary and covariate effects on PON1 arylesterase activity. Five dietary components, cholesterol (P < 2.0 × 10(-16)), alcohol (P = 8.51 × 10(-8)), vitamin C (P = 7.97 × 10(-5)), iron (P = 0.0026), and folic acid (0.037) were independently predictive of PON1 activity. Dietary cholesterol was positively associated and predicted 5.5% of PON1 activity, second in variance explained. This study presents a novel finding of dietary cholesterol, iron, and folic acid predicting PON1 activity in humans and confirms prior reported associations, including that with vitamin C. Identifying and understanding environmental factors that affect PON1 activity is necessary to understand its role and that of HDL in human disease.

Analysis of recently identified dyslipidemia alleles reveals two loci that contribute to risk for carotid artery disease.

BACKGROUND: Genome-wide association studies have identified numerous single nucleotide polymorphisms (SNPs) affecting high density lipoprotein (HDL) or low density lipoprotein (LDL) cholesterol levels; these SNPs may contribute to the genetic basis of vascular diseases. RESULTS: We assessed the impact of 34 SNPs at 23 loci on dyslipidemia, key lipid sub-phenotypes, and severe carotid artery disease (CAAD) in a case-control cohort. The effects of these SNPs on HDL and LDL were consistent with those previously reported, and we provide unbiased estimates of the percent variance in HDL (3.9%) and LDL (3.3%) explained by genetic risk scores. We assessed the effects of these SNPs on HDL subfractions, apolipoprotein A-1, LDL buoyancy, apolipoprotein B, and lipoprotein (a) and found that rs646776 predicts apolipoprotein B level while rs2075650 predicts LDL buoyancy. Finally, we tested the role of these SNPs in conferring risk for ultrasonographically documented CAAD stenosis status. We found that two loci, chromosome 1p13.3 near CELSR2 and PSRC1 which contains rs646776, and 19q13.2 near TOMM40 and APOE which contains rs2075650, harbor risk alleles for CAAD. CONCLUSION: Our analysis of 34 SNPs contributing to dyslipidemia at 23 loci suggests that genetic variation in the 1p13.3 region may increase risk of CAAD by increasing LDL particle number, whereas variation in the 19q13.2 region may increase CAAD risk by promoting formation of smaller, denser LDL particles.

Effects of dietary components on high-density lipoprotein measures in a cohort of 1,566 participants.

BACKGROUND: Recent data suggest that an increased level of high-density lipoprotein cholesterol (HDL-C) is not causally protective against heart disease, shifting focus to other sub-phenotypes of HDL. Prior work on the effects of dietary intakes has focused largely on HDL-C. The goal of this study was to identify the dietary intakes that affect HDL-related measures: HDL-C, HDL-2, HDL-3, and apoA1 using data from a carotid artery disease case-control cohort. METHODS: A subset of 1,566 participants with extensive lipid phenotype data completed the Harvard Standardized Food Frequency Questionnaire to determine their daily micronutrient intake over the past year. Stepwise linear regression was used to separately evaluate the effects of dietary covariates on adjusted levels of HDL-C, HDL-2, HDL-3, and apoA1. RESULTS: Dietary folate intake was positively associated with HDL-C (p = 0.007), HDL-2 (p = 0.0011), HDL-3 (p = 0.0022), and apoA1 (p = 0.001). Alcohol intake and myristic acid (14:0), a saturated fat, were each significantly associated with increased levels of all HDL-related measures studied. Dietary carbohydrate and iron intake were significantly associated with decreased levels of all HDL-related measures. Magnesium intake was positively associated with HDL-C, HDL-2, and HDL-3 levels, but not apoA1 levels, while vitamin C was only associated with apoA1 levels. Dietary fiber and protein intake were both associated with HDL-3 levels alone. CONCLUSIONS: This study is the first to report that dietary folate intake is associated with HDL-C, HDL-2, HDL-3, and apoA1 levels in humans. We further identify numerous dietary intake associations with apoA1, HDL-2, and HDL-3 levels. Given the shifting focus away from HDL-C, these data will prove valuable for future epidemiologic investigation of the role of diet and multiple HDL phenotypes in heart disease.

HDL-3 is a superior predictor of carotid artery disease in a case-control cohort of 1725 participants.

BACKGROUND: Recent data suggest that high-density lipoprotein cholesterol (HDL-C) levels are likely not in the causative pathway of atheroprotection, shifting focus from HDL-C to its subfractions and associated proteins. This study's goal was to determine which HDL phenotype was the better predictor of carotid artery disease (CAAD). METHODS AND RESULTS: HDL-2 and HDL-3 were measured in 1725 participants of European ancestry in a prevalent case-control cohort study of CAAD. Stratified analyses were conducted for men (n=1201) and women (n=524). Stepwise linear regression was used to determine whether HDL-C, HDL-2, HDL-3, or apolipoprotein A1 was the best predictor of CAAD, while adjusting for the confounders of censored age, diabetes, and current smoking status. In both men and women, HDL-3 was negatively associated with CAAD (P=0.0011 and 0.033 for men and women, respectively); once HDL-3 was included in the model, no other HDL phenotype was significantly associated with CAAD. Addition of paraoxonase 1 activity to the aforementioned regression model showed a significant and independent (of HDL-3) association with CAAD in men (P=0.001) but not in the smaller female subgroup. CONCLUSIONS: This study is the first to contrast the associations of HDL-2 and HDL-3 with CAAD. We found that HDL-3 levels were more predictive of CAAD status than HDL-2, HDL-C, or apolipoprotein A1. In addition, for men, paraoxonase 1 activity improved the overall model prediction for CAAD independently and additively with HDL-3 levels. Further investigation into the molecular mechanisms through which HDL-3 is associated with protection from CAAD is warranted.

Additional Common Polymorphisms in the PON Gene Cluster Predict PON1 Activity but Not Vascular Disease.

Background. Paraoxonase 1 (PON1) enzymatic activity has been consistently predictive of cardiovascular disease, while the genotypes at the four functional polymorphisms at PON1 have not. The goal of this study was to identify additional variation at the PON gene cluster that improved prediction of PON1 activity and determine if these variants predict carotid artery disease (CAAD). Methods. We considered 1,328 males in a CAAD cohort. 51 tagging single-nucleotide polymorphisms (tag SNPs) across the PON cluster were evaluated to determine their effects on PON1 activity and CAAD status. Results. Six SNPs (four in PON1 and one each in PON2/3) predicted PON1 arylesterase (AREase) activity, in addition to the four previously known functional SNPs. In total, the 10 SNPs explained 30.1% of AREase activity, 5% of which was attributable to the six identified predictive SNPs. We replicate rs854567 prediction of 2.3% of AREase variance, the effects of rs3917510, and a PON3 haplotype that includes rs2375005. While AREase activity strongly predicted CAAD, none of the 10 SNPs predicting AREase predicted CAAD. Conclusions. This study identifies new genetic variants that predict additional PON1 AREase activity. Identification of SNPs associated with PON1 activity is required when evaluating the many phenotypes associated with genetic variation near PON1.

The G protein-coupled receptor repertoires of human and mouse.

Diverse members of the G protein-coupled receptor (GPCR) superfamily participate in a variety of physiological functions and are major targets of pharmaceutical drugs. Here we report that the repertoire of GPCRs for endogenous ligands consists of 367 receptors in humans and 392 in mice. Included here are 26 human and 83 mouse GPCRs not previously identified. A direct comparison of GPCRs in the two species reveals an unexpected level of orthology. The evolutionary preservation of these molecules argues against functional redundancy among highly related receptors. Phylogenetic analyses cluster 60% of GPCRs according to ligand preference, allowing prediction of ligand types for dozens of orphan receptors. Expression profiling of 100 GPCRs demonstrates that most are expressed in multiple tissues and that individual tissues express multiple GPCRs. Over 90% of GPCRs are expressed in the brain. Strikingly, however, the profiles of most GPCRs are unique, yielding thousands of tissue- and cell-specific receptor combinations for the modulation of physiological processes.