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Riboflavin is a feed additive, food additive, and clinical drug, with a significant annual demand of nearly 8000 tons. Fermentation using recombinant Bacillus subtilis is currently one of the most important industrial production method for riboflavin. First, a suitable medium was selected and the expression of the ureABC operon was modified. The ykgB gene was overexpressed in B. subtilis RX10, the production of the derivative strain RX20 was increased to 4.61 g l-1 riboflavin, and the yield was increased to 52 mg riboflavin g-1 glucose. The relative transcription level of pyr operon in RX20 was reduced to 71%, the production of the derivative strain RX21 was increased to 5.82 g l-1 riboflavin, and the yield was 76 mg riboflavin g-1 glucose. The start codon of the pyrE gene in RX21 was modified to "TTG", the production of the derivative strain RX22 was increased to 7.01 g l-1 riboflavin, and the yield was 89 mg riboflavin g-1 glucose. These results indicated that overexpression of the ykgB gene and reduction of the metabolic flux of de novo synthesis of pyrimidine nucleotides were beneficial to the synthesis of riboflavin.

As an intermediate in drug synthesis, uridine has practical applications in the pharmaceutical field. Bacillus subtilis is used as a host to boost uridine yield by manipulating its uridine biosynthesis pathway. In this study, we engineered a high-uridine-producing strain of B. subtilis by modifying its metabolic pathways in vivo. Overexpression of the aspartate ammonia-lyase (ansB) gene increased the relative transcriptional level of ansB in B. subtilis TD320 by 13·18 times and improved uridine production to 15·13 g l−1 after 72-h fermentation. Overexpression of the putative 6-phosphogluconolactonase (ykgB) gene increased uridine production by the derivative strain TD325 to 15·43 g l−1. Reducing the translation of the amido phosphoribosyl transferase (purF) gene and inducing expression of the subtilisin E (aprE) gene resulted in a 1·99-fold increase in uridine production after 24 h shaking. Finally, uridine production in the optimal strain B. subtilis TD335, which exhibited reduced urease expression, reached 17·9 g l−1 with a yield of 314 mg of uridine g−1 glucose. To our knowledge, this is the first study to obtain high-yield uridine-producing B. subtilis in a medium containing only three components (80 g l−1 glucose, 20 g l−1 yeast powder, and 20 g l−1 urea).

Supplemental Digital Content is available in the text., Background: Familial hypercholesterolemia (FH) is a common autosomal codominant genetic disorder, which causes elevated levels of low-density lipoprotein cholesterol (LDL-C) and increased risk of premature atherosclerotic cardiovascular disease (ASCVD). Even among individuals with monogenic FH, there is substantial interindividual variability in LDL-C levels and risk of ASCVD. We assessed the influence of an LDL-C polygenic score on levels of LDL-C and risk of ASCVD for individuals with monogenic FH. Methods: We constructed a weighted LDL-C polygenic score, composed of 28 single-nucleotide variants, for individuals with monogenic FH from the British Columbia FH (n=262); Nutrition, Metabolism and Atherosclerosis Clinic (n=552); and UK Biobank cohorts (n=306). We assessed the association between LDL-C polygenic score with LDL-C levels and ASCVD risk using linear regression and Cox-proportional hazard models, respectively. ASCVD was defined as myocardial infarction, coronary or carotid revascularization, transient ischemic attack, or stroke. The results from individual cohorts were combined in fixed-effect meta-analyses. Results: Levels of LDL-C were significantly associated with LDL-C polygenic score in the Nutrition, Metabolism and Atherosclerosis Clinic cohort, UK Biobank cohort, and in the meta-analysis (β [95% CI]=0.13 [0.072–0.19] per a 20% increase in LDL-C polygenic score percentile, P

Background: Familial hypercholesterolemia (FH) is an inherited condition of elevated serum low-density lipoprotein (LDL) cholesterol leading to premature coronary heart disease. We evaluated whether FH mutations are independently associated with the development of myocardial infarction (MI), after adjusting for LDL cholesterol level and clinical risk factors. Methods: In 182 unrelated patients from different families referred with clinically suspected FH, targeted next-generation DNA sequencing was performed on 73 lipid-related genes and 178 single nucleotide polymorphisms, at 300-times mean read depth, to identify monogenic mutations and high-risk single nucleotide polymorphisms. Results: Pathogenic FH mutations were identified in 27% of patients. Patients with mutations, compared with those without, were 12 years younger when referred to the lipid clinic (P < 0.001) and had higher baseline and post-treatment LDL cholesterol by 1.11 mmol/L (P

Background: Coronary artery disease (CAD) risk traditionally has been assessed using clinical risk factors. We evaluated whether molecular genetic markers for CAD risk could add information to traditional variables. Methods: We developed a false discovery rate 267-marker genetic risk score (FDR267) from markers that were significantly associated with CAD in the UK Biobank cohort meta-analysis. FDR267 was tested in the Atherosclerosis Risk in Communities cohort using logistic regression and Cox proportional hazards analyses in the European and African American groups. Results: Our genetic risk score (FDR267) was associated with a 1.45 (95% confidence interval, 1.39-1.51) increase in odds ratio and a 1.32 (95% confidence interval, 1.26-1.38) increase in hazard ratio per standard deviation of the score. The score modestly improved the area under the curve (AUC) statistic when added to a clinical model (ΔAUC = 0.0112, P = 0.0002). FDR267 predicted incident CAD (C-index = 0.60), although it did not improve on clinical risk factors (ΔAUC = 0.0159, P = 0.0965). Individuals in the top quintile of FDR267 genetic risk were at approximately 2-fold increased risk compared with the bottom quintile, which is comparable to risk associated with self-reported family history. The performance of FDR267 was less robust in the African American sample. Conclusions: FDR267 is significantly associated with CAD in the European sample, with an effect size comparable to self-reported family history. FDR267 discriminated between individuals with and without CAD, but did not improve CAD risk prediction over clinical variables. FDR267 was less predictive of CAD risk in African Americans. Résumé: Introduction: L’évaluation du risque de maladie coronarienne (MC) a traditionnellement reposé sur les facteurs de risque cliniques. Nous avons évalué si les marqueurs génétiques moléculaires de risque de MC pourraient servir de complément aux variables traditionnelles. Méthodes: Nous avons élaboré un taux de fausses découvertes (FDR pour false discovery rate) du score de risque génétique du marqueur 267 (FDR267) provenant des marqueurs qui étaient associés de manière significative à la MC dans la méta-analyse de cohortes de la UK Biobank. Le FDR267 a été testé dans la cohorte du Atherosclerosis Risk in Communities à l’aide de la régression logistique et des analyses selon le modèle à risques proportionnels de Cox dans des groupes européens et afro-américains. Résultats: Notre score de risque génétique (FDR267) a été associé à une augmentation de 1,45 (intervalle de confiance [IC] à 95 %, 1,39-1,51) du rapport des cotes et d’une augmentation de 1,32 (IC à 95 %, 1,26-1,38) du risque relatif par l’écart-type des scores. Le score a modestement amélioré l’aire sous la courbe (ASC) lorsqu’il a été ajouté à un modèle clinique (ΔASC = 0,0112, P = 0,0002). Le FDR267 a prédit les nouveaux cas de MC (C-index [indice de concordance] = 0,60), mais il n’a pas amélioré les facteurs de risque cliniques (ΔASC = 0,0159, P = 0,0965). Les individus dans le quintile supérieur du risque génétique du FDR267 ont montré un risque accru d’environ 2 fois par rapport au quintile inférieur, soit un risque comparable au risque associé aux antécédents familiaux auto-rapportés. La performance du FDR267 s’est révélée moins robuste chez les Afro-Américains. Conclusions: Le FDR267 est associé de manière significative à la MC dans l’échantillon d’Européens et a une taille de l’effet comparable aux antécédents familiaux auto-rapportés. Le FDR267 a fait la discrimination entre les individus atteints ou non atteints de MC, mais n’a pas amélioré la prédiction du risque de MC par rapport aux variables cliniques. Le FDR267 a moins bien prédit le risque de MC chez les Afro-Américains.

A 41-year-old male Chinese patient had an incidental finding of elevated serum creatine kinase (4000 U/L, normal range 2–200 U/L) during a routine physical examination 3 years ago, which fluctuated...

Background In 2013, our laboratory designed a targeted sequencing panel, “LipidSeq”, to study the genetic determinants of dyslipidemia and metabolic disorders. Over the last 6 years, we have analyzed 3262 patient samples obtained from our own Lipid Genetics Clinic and international colleagues. Here, we highlight our findings and discuss research benefits and clinical implications of our panel. Methods LipidSeq targets 69 genes and 185 single-nucleotide polymorphisms (SNPs) either causally related or associated with dyslipidemia and metabolic disorders. This design allows us to simultaneously evaluate monogenic—caused by rare single-nucleotide variants (SNVs) or copy-number variants (CNVs)—and polygenic forms of dyslipidemia. Polygenic determinants were assessed using three polygenic scores, one each for low-density lipoprotein cholesterol, triglyceride, and high-density lipoprotein cholesterol. Results Among 3262 patient samples evaluated, the majority had hypertriglyceridemia (40.1%) and familial hypercholesterolemia (28.3%). Across all samples, we identified 24,931 unique SNVs, including 2205 rare variants predicted disruptive to protein function, and 77 unique CNVs. Considering our own 1466 clinic patients, LipidSeq results have helped in diagnosis and improving treatment options. Conclusions Our LipidSeq design based on ontology of lipid disorders has enabled robust detection of variants underlying monogenic and polygenic dyslipidemias. In more than 50 publications related to LipidSeq, we have described novel variants, the polygenic nature of many dyslipidemias—some previously thought to be primarily monogenic—and have uncovered novel mechanisms of disease. We further demonstrate several tangible clinical benefits of its use.

HDL cholesterol (HDL-C) remains a superior biochemical predictor of CVD risk, but its genetic basis is incompletely defined. In patients with extreme HDL-C concentrations, we concurrently evaluated the contributions of multiple large- and small-effect genetic variants. In a discovery cohort of 255 unrelated lipid clinic patients with extreme HDL-C levels, we used a targeted next-generation sequencing panel to evaluate rare variants in known HDL metabolism genes, simultaneously with common variants bundled into a polygenic trait score. Two additional cohorts were used for validation and included 1,746 individuals from the Montréal Heart Institute Biobank and 1,048 individuals from the University of Pennsylvania. Findings were consistent between cohorts: we found rare heterozygous large-effect variants in 18.7% and 10.9% of low- and high-HDL-C patients, respectively. We also found common variant accumulation, indicated by extreme polygenic trait scores, in an additional 12.8% and 19.3% of overall cases of low- and high-HDL-C extremes, respectively. Thus, the genetic basis of extreme HDL-C concentrations encountered clinically is frequently polygenic, with contributions from both rare large-effect and common small-effect variants. Multiple types of genetic variants should be considered as contributing factors in patients with extreme dyslipidemia.

Density (ρ) and viscosity (η) of glycine (c = 0.02–0.22 mol dm−3) in aqueous urea (c = 0.5, 1.5, and 3.0 mol dm−3) solutions were measured at 298.15 K. Experimental density data has been used to calculate apparent molar volumes (φv) of glycine in aqueous and aqueous-urea solutions at 298.15 K. The dependence of apparent molar volumes on concentration of glycine was fitted to the Massons relation and apparent molar volume of glycine at infinite dilution (partial molar volume, φv0) was determined graphically. The partial molar volumes of transfer (Δtrφv0) of glycine at infinite dilution from pure water to aqueous-urea solutions at 298.15 K were calculated and interpreted in terms of various interactions and structural fittings in studied solutions. The relative viscosity data has been analyzed by Jones-Dole relation and viscosity B-coefficients were determined graphically. Viscosity B-coefficient of transfer (ΔB) was also calculated and compared with Δtrφv0.

Rationale: Although HDL-C levels are known to have a complex genetic basis, most studies have focused solely on identifying rare variants with large phenotypic effects to explain extreme HDL-C phenotypes. Objective: Here we concurrently evaluate the contribution of both rare and common genetic variants, as well as large-scale copy number variations (CNVs), towards extreme HDL-C concentrations. Methods: In clinically ascertained patients with low ( N =136) and high ( N =119) HDL-C profiles, we applied our targeted next-generation sequencing panel (LipidSeq TM ) to sequence genes involved in HDL metabolism, which were subsequently screened for rare variants and CNVs. We also developed a novel polygenic trait score (PTS) to assess patients’ genetic accumulations of common variants that have been shown by genome-wide association studies to associate primarily with HDL-C levels. Two additional cohorts of patients with extremely low and high HDL-C (total N =1,746 and N =1,139, respectively) were used for PTS validation. Results: In the discovery cohort, 32.4% of low HDL-C patients carried rare variants or CNVs in primary ( ABCA1 , APOA1 , LCAT ) and secondary ( LPL , LMF1 , GPD1 , APOE ) HDL-C–altering genes. Additionally, 13.4% of high HDL-C patients carried rare variants or CNVs in primary ( SCARB1 , CETP , LIPC , LIPG ) and secondary ( APOC3 , ANGPTL4 ) HDL-C–altering genes. For polygenic effects, patients with abnormal HDL-C profiles but without rare variants or CNVs were ~2-fold more likely to have an extreme PTS compared to normolipidemic individuals, indicating an increased frequency of common HDL-C–associated variants in these patients. Similar results in the two validation cohorts demonstrate that this novel PTS successfully quantifies common variant accumulation, further characterizing the polygenic basis for extreme HDL-C phenotypes. Conclusions: Patients with extreme HDL-C levels have various combinations of rare variants, common variants, or CNVs driving their phenotypes. Fully characterizing the genetic basis of HDL-C levels must extend to encompass multiple types of genetic determinants—not just rare variants—to further our understanding of this complex, controversial quantitative trait.

Aims Activation of vascular GPER has been linked to vasodepressor effects in animals. However, the significance of GPER regulation on chronic blood pressure control in humans is unknown. Methods To examine this question we determined the functional significance of expression of a common missense single nucleotide variant of GPER, P16L in vascular smooth muscle cells, and its association with blood pressure in humans. Further, to validate the importance of carrying GPER P16L in the development of hypertension we assessed allele frequency in a cohort of hard-to-treat hypertensive patients referred to a tertiary care clinic. Results Expression of the GPER P16L variant (V) vs. wild type (WT) in rat aortic vascular smooth muscle cells, was associated with a significant decrease in G1 (1 μm, a GPER agonist)-mediated ERK phosphorylation (slope of the function of G1-stimulated ERK phosphorylation: GPER content WT: 16.2, 95% CI 9.9, 22.6; V: 5.0, 95% CI 1.0, 9.0; P < 0.005) and apoptosis (slope of the function of G1-stimulated apoptosis: GPER content: WT: 4.4, 95% CI: 3.4, 5.4; V: 2.5, 95% CI 1.6, 2.3 P < 0.005). Normotensive female subjects, but not male subjects, carrying this hypofunctional variant (allele frequency 22%) have increased blood pressure [mean arterial pressure: P16/P16: 80 ± 1 mmHg (n = 204) vs. P16L carriers: 82 ± 1 mmHg (n = 127), 95% CI for difference: 0.6, 4.0 mmHg, P < 0.05], [systolic blood pressure: P16/P16: 105 ± 1 mmHg vs. P16L carriers: 108 ± 1 mmHg, 95% CI for difference:1.0, 5.1 mmHg, P < 0.05], [diastolic blood pressure: P16/P16: 66 ± 0.5 mmHg vs. P16L carriers 68 ± 0.7, 95% CI for difference: 0.2, 3.6 mmHg, P < 0.05]. Further, the P16L allele frequency was almost two-fold higher in female vs. male hypertensive patients (31% vs. 16%, allele ratio 0.5, 95% CI 0.32, 0.76, P < 0.05). Conclusions The common genetic variant, GPER P16L, is hypofunctional and female carriers of this allele have increased blood pressure. There was an increased prevalence in a population of hard-to-treat hypertensive female patients. Cumulatively, these data suggest that in females, impaired GPER function might be associated with increased blood pressure and risk of hypertension.

Objective Depressed high-density lipoprotein cholesterol (HDL-C) is prevalent the Middle East and North Africa. Some studies have documented associations between HDL-C and several single nucleotide polymorphisms (SNPs) in candidate gene polymorphisms. Methods We investigated the associations between SNP genotypes and HDL-C levels in Iranian students, aged 10–18 years. Genotyping was performed in 750 randomly selected participants among those with low HDL-C levels (below 5th percentile), intermediate HDL-C levels (5–95th) and high HDL-C levels (above the 95th percentile). Minor allele frequencies (MAFs) of the SNPs of interest were compared between the three HDL-C groups. Results The vast majority of pairwise comparisons of MAFs between HDL-C groups were significant. Pairwise comparisons between low and high HDL-C groups showed significant between-group differences in MAFs for all SNPs, except for APOC3 rs5128. Pairwise comparisons between low and intermediate HDL-C groups showed significant between-group differences in MAFs for all SNPs, except for APOC3 rs5128 and APOA1 rs2893157. Pairwise comparisons between intermediate and high HDL-C groups showed significant between-group differences in MAFs for all SNPs, except for ABCA1 APOC3 rs5128 and APOA1 rs2893157. After adjustment for confounding factors, including age, sex, body mass index, low physical activity, consumption of saturated fats, and socioeconomic status, ABCA1 r1587K and CETP A373P significantly increased the risk of depressed HDL-C, and CETP Taq1 had a protective role. Conclusion This study replicated several associations between HDL-C levels and candidate gene SNPs from genome-wide associations with HDL-C in Iranians from the pediatric age group.

Background The loss of patent protection for proprietary statins offers affordability advantages to payers, but some clinicians still question the efficacy of generic formulations in real-world clinical applications. Methods In this retrospective cohort study, we examined the effects of generic atorvastatin substitution on relevant biochemical parameters in 85 dyslipidemic patients who had been previously maintained on stable doses of proprietary atorvastatin from 2009 to 2011. For comparison, we studied 143 patients who were continuously prescribed stable doses of rosuvastatin, which was only available in its proprietary formulation over the same time period. Results We found that substitution of generic for proprietary atorvastatin was not associated with significant changes in plasma levels of total or low-density lipoprotein cholesterol, or triglycerides, but was associated with a small but significant increase in high-density lipoprotein cholesterol. Plasma levels of aspartate aminotransferase and creatine kinase were also unchanged. Additionally, the changeover to generic atorvastatin was not associated with increased switching to another statin or more frequent changes in other lipid-lowering medications compared with the proprietary rosuvastatin group. Conclusions Substituting generic for proprietary atorvastatin in lipid clinic patients was not associated with significant changes in efficacy, adverse events, or patient management.

Objective— Next-generation sequencing technology is transforming our understanding of heterozygous familial hypercholesterolemia, including revision of prevalence estimates and attribution of polygenic effects. Here, we examined the contributions of monogenic and polygenic factors in patients with severe hypercholesterolemia referred to a specialty clinic. Approach and Results— We applied targeted next-generation sequencing with custom annotation, coupled with evaluation of large-scale copy number variation and polygenic scores for raised low-density lipoprotein cholesterol in a cohort of 313 individuals with severe hypercholesterolemia, defined as low-density lipoprotein cholesterol >5.0 mmol/L (>194 mg/dL). We found that (1) monogenic familial hypercholesterolemia–causing mutations detected by targeted next-generation sequencing were present in 47.3% of individuals; (2) the percentage of individuals with monogenic mutations increased to 53.7% when copy number variations were included; (3) the percentage further increased to 67.1% when individuals with extreme polygenic scores were included; and (4) the percentage of individuals with an identified genetic component increased from 57.0% to 92.0% as low-density lipoprotein cholesterol level increased from 5.0 to >8.0 mmol/L (194 to >310 mg/dL). Conclusions— In a clinically ascertained sample with severe hypercholesterolemia, we found that most patients had a discrete genetic basis detected using a comprehensive screening approach that includes targeted next-generation sequencing, an assay for copy number variations, and polygenic trait scores.

HDL cholesterol (HDL-C) levels strongly associate with cardiovascular disease risk, and as a complex trait, are influenced by genetic and environmental factors. Extreme HDL-C concentrations are largely genetically determined; monogenic disorders of HDL-C have been well-characterized, including the primary candidate genes driving each extreme phenotype, which typically show autosomal recessive or co-dominant inheritance. Within genes causing syndromes of both HDL-C extremes, numerous disease-causing variants have been identified and functionally validated. In a unique cohort of patients with extreme HDL-C profiles ( N =255), we applied our targeted next-generation sequencing panel LipidSeq TM , which is designed for clinical re-sequencing of genes associated with dyslipidemia and other metabolic disorders. We found that 20.6% and 11.8% of low ( N =136) and high ( N =119) HDL-C patients, respectively, carry heterozygous, large-effect mutations in pertinent genes explaining their phenotypes. To further characterize the genetic variation contributing to HDL-C levels, we next investigated the integrated polygenic contribution from multiple small-effect genetic variants using a polygenic trait score (PTS). We developed two scores to assess an individual’s burden of small-effect variants: one each for lowering and raising HDL-C levels. As a whole, low HDL-C patients had a significantly greater mean PTS for low HDL-C than normolipidemic controls ( P P

Objectives Maturity-onset diabetes of the young (MODY) is the most common form of monogenic diabetes, reportedly accounting for 2% to 5% of all cases of diabetes. In samples from Canadian patients referred for molecular genetic confirmation of a clinically suspected MODY, we determined the prevalence of likely disease-causing DNA variants in known MODY genes. Methods Between 1999 and 2015, our centre received requests from colleagues for DNA sequencing of 96 samples from unrelated Canadian patients with clinically suspected MODY. Prior to 2012, we used Sanger sequencing, and since 2012 we have used targeted next-generation sequencing. Results Of 96 samples received, 39 (40.6%) had a likely rare causal variant in 1 of 8 known MODY genes. Of these, 20 (51.3%) and 19 (48.7%) were diagnosed by Sanger and targeted next-generation sequencing, respectively. The 39 mutation-positive samples had 1 of 39 rare variants, of which the majority were in genes encoding either glucokinase ( GCK , or MODY2) or hepatocyte nuclear factor 1-alpha ( HNF1A , or MODY3). Furthermore, 12 (30.8%) of the detected rare variants had been unreported previously but were likely to have been clinically significant according to standard bioinformatic methods. An additional 6 samples had rare variants in MODY genes that were of uncertain clinical significance. Conclusions The findings suggest that clinical suspicion for MODY has a diagnostic yield of ~40% at the molecular level. Confirmatory genetic testing in patients suspected to have MODY allows for definitive diagnoses which, in turn, may guide management and provide rationales for screening other family members presymptomatically.

Genome-wide association studies (GWAS) for plasma lipid levels have mapped numerous genomic loci, with each region often containing many protein-coding genes. Targeted re-sequencing of exons is a strategy to pinpoint causal variants and genes. We performed solution-based hybrid selection of 9008 exons at 939 genes within 95 GWAS loci for plasma lipid levels and sequenced using next-generation sequencing technology individuals with extremely high as well as low to normal levels of low-density lipoprotein cholesterol (LDL-C, n = 311; mean low = 71 mg/dl versus high = 241 mg/dl), triglycerides (TG, n = 308; mean low = 75 mg/dl versus high = 1938 mg/dl), and high-density lipoprotein cholesterol (HDL-C, n = 684; mean low = 32 mg/dl versus high = 102 mg/dl). We identified 15,002 missense, nonsense, or splice site variants with a frequency

Background— Rare variant accumulation studies can implicate genes in disease susceptibility when a significant burden is observed in patients versus control subjects. Such analyses might be particularly useful for candidate genes that are selected based on experiments other than genome-wide association studies (GWAS). We sought to determine whether rare variants in non-GWAS candidate genes identified from mouse models and human mendelian syndromes of hypertriglyceridemia (HTG) accumulate in patients with polygenic adult-onset HTG. Methods and Results— We resequenced protein coding regions of 3 genes with established roles ( APOC2, GPIHBP1 , LMF1 ) and 2 genes recently implicated ( CREB3L3 and ZHX3 ) in TG metabolism. We identified 41 distinct heterozygous rare variants, including 29 singleton variants, in the combined sample; in total, we observed 47 rare variants in 413 HTG patients versus 16 in 324 control subjects (odds ratio=2.3; P =0.0050). Post hoc assessment of genetic burden in individual genes using 3 different tests suggested that the genetic burden was most prominent in the established genes LMF1 and APOC2 , and also in the recently identified CREB3L3 gene. Conclusions— These extensive resequencing studies show a significant accumulation of rare genetic variants in non-GWAS candidate genes among patients with polygenic HTG, and indicate the importance of testing specific hypotheses in large-scale resequencing studies.

Background Familial chylomicronemia syndrome (FCS) is an ultra-rare phenotype that is usually caused by biallelic mutations in the LPL gene encoding lipoprotein lipase, or less often in APOC2, APOA5, LMF1 , or GPIHBP1 genes encoding cofactors or interacting proteins. Objectives We evaluated baseline phenotypes among FCS participants in a phase 3 randomized placebo-controlled trial of volanesorsen (NCT02211209). Methods Baseline clinical, fasting, and postfat load metabolic markers were assessed. Targeted next-generation DNA sequencing plus custom bioinformatics was used to genotype subjects. Results Among 52 FCS individuals, 41 had biallelic LPL gene mutations ( LPL- FCS patients): 82%, 7%, and 11% were missense, nonsense, and splicing variants, respectively. Eleven individuals had non- LPL -FCS; 2 had mutations in APOA5 , 5 in GPIHBP1 , and 1 each in LMF1 and APOC2 genes, respectively. Two other individuals were double heterozygotes, each with 1 normal LPL allele. All subjects had extremely high triglycerides (TGs) and chylomicrons, but very low levels of other lipoproteins. Compared with LPL -FCS individuals, non- LPL -FCS individuals were very similar for most traits, but had significantly higher postheparin LPL activity, higher 4-hour postprandial insulin and C-peptide levels; and higher low-density lipoprotein cholesterol levels. In non- LPL -FCS individuals compared to those with LPL -FCS, there were also nonsignificant trends toward lower levels of total and chylomicron TGs, lower 4-hour postprandial chylomicron TG levels, and higher very-low-density lipoprotein TG levels. Conclusion Thus, LPL FCS and non- LPL FCS are largely phenotypically similar. However, LPL FCS patients have lower postheparin LPL activity and a trend toward higher TGs, whereas low-density lipoprotein cholesterol was higher in non- LPL -FCS patients.

Circulating matrix metalloproteinase (MMP) levels may correlate with diabetic complications. Whether they are changed in early diabetic cardiomyopathy is not known and was examined in this study. TIMP-1 and collagen degradation products were also measured. Results from subjects with and without diastolic dysfunction were compared with those obtained for patients with varying stages of diabetic renal disease. Patients with type 2 diabetes with or without diastolic dysfunction with varying degrees of renal disease were recruited for this study. Age-matched non-diabetic subjects served as controls. MMPs (-1, -3 and -7) and TIMP-1 were measured by ELISA, MMP-2 and -9 by zymography and collagen degradation products by radioimmunoassay. Differences in the pattern of MMPs/TIMPs and collagen degradation products were observed. The most consistent change was in totalMMP-7, which was increased in those with diastolic dysfunction and those with macroalbuminuria. MMP-7 correlated with cardiac function (p

Numerous single nucleotide polymorphisms (SNPs) have been found in recent genome wide association studies (GWAS) to be associated with subtle plasma triglyceride (TG) variation in normolipidemic subjects. However, since these GWAS did not specifically evaluate patients with rare disorders of lipoprotein metabolism--'hyperlipoproteinemia' (HLP)--it remains largely unresolved whether any of these SNP determinants of modest physiological changes in TG are necessarily also determinants of most HLP phenotypes. To address this question, we evaluated 28 TG-associated SNPs from GWAS in 386 unrelated adult patients with one of five Fredrickson phenotypes (HLP types 2A, 2B, 3, 4 and 5) and 242 matched normolipidemic controls. We found that several SNPs associated with TG in normolipidemic samples, including APOA5 p.S19W and -1131T>C, TRIB1 rs17321515, TBL2 rs17145738, GCKR rs780094, GALNT2 rs4846914 and ANGPTL3 rs12130333, were significantly associated with HLP types 2B, 3, 4 and 5. The findings indicate that: (i) the TG-associated Fredrickson HLP types 2B, 3, 4 and 5 are polygenic traits; (ii) these Fredrickson HLP types share numerous genetic determinants among themselves; and (iii) genetic determinants of modest TG variation in normolipidemic population samples also underlie--to an apparently even greater degree--susceptibility to these rare HLP phenotypes. Thus, the TG-associated Fredrickson HLP types 2B, 3, 4 and 5, although historically considered to be distinct are actually complex traits sharing among them several common genetic determinants seen in GWAS of normolipidemic population samples.

Carnitine palmitoyltransferase IA, encoded by CPT1A, is a key regulator of fatty acid metabolism. Previously, a loss-of-function mutation, namely, c.1436 C-->T (p.P479L), was reported in CPT1A in the homozygous state in Canadian aboriginal male with presumed CPT1A deficiency. To determine the population frequency of this variant, we determined CPT1A p.P479L genotypes in 1111 Greenland Inuit. Associations between genotype and variation in plasma total cholesterol, triglycerides, LDL, HDL, apolipoprotein (apo) B, and apoA-I was also investigated. We found the L479 allele occurs at a high frequency in this sample (0.73), while it was completely absent in 285 nonaboriginal samples. This suggests that the original proband's symptoms were not likely due to the CPT1A p.P479L mutation because it is very common in Inuit and because symptoms suggesting CPT1A deficiency have not been reported in any carrier subsequently studied. However, CPT1A p.P479L was associated with elevated plasma HDL and apoA-I levels. The association with increased levels of HDL and apoA-I suggest that the polymorphism might protect against atherosclerosis.

Aim: Diabetic cardiomyopathy is an increasingly recognized entity. The pathogenic factors that may contribute to its development, especially the earliest changes of diastolic dysfunction (DD), have not been clearly defined. Microvessel dysfunction and upregulation of profibrotic growth factors have been described as possible causes. The aim of this study was therefore to determine whether microvascular dysfunction and/or upregulation of the profibrotic connective tissue growth factor (CTGF) are associated with subclinical DD in subjects with type 2 diabetes. Methods: Forty subjects with type 2 diabetes and 20 age-matched non-diabetic controls, all of whom had no clinical evidence of ischaemic heart disease, cardiac failure or echo evidence of systolic ventricular dysfunction, were recruited. Microvascular function was measured by laser Doppler velocimetry, with examination of endothelium-dependent increase in blood flow following iontophoresis of acetylcholine (ACh) and endothelium-independent increase in blood flow in response to the nitric oxide donor sodium nitroprusside (SNP). CTGF levels were determined by Western immunoblotting. Results: DD determined on the basis of traditional echocardiographic criteria was similar in diabetic subjects compared with controls (28 vs. 20%, p = 0.5). Using left ventricular myocardial tissue Doppler-based indices for DD, the E/E′ and the E′/A′ ratios (where E is the flow related to early ventricular filling and E′ and A′ are early and late diastolic velocities, respectively) in diabetic subjects revealed evidence of more DD than controls (p = 0.046 and p = 0.007 respectively) . Comparing controls with no DD by conventional echocardiographic criteria (Group I), diabetes and no DD (Group II) and diabetes with DD (Group III), there was a significant trend in reduction of both endothelium-dependent (ACh fold change; p = 0.04) and endothelium-independent (SNP fold change; p = 0.0004) blood flow across the groups. The ACh and SNP responses, however, were not correlated significantly with quartiles of the E/E′ ratio or the E′/A′ ratio. CTGF plasma levels did not differ across the groups and CTGF did not correlate with parameters of microvascular function. Conclusions: This study indicates that while there is a significant association between DD and measures of microvascular function, the relationship between endothelial dysfunction, CTGF and subtle measures of DD is not strong. Other factors are therefore likely to play an important role in the early pathogenesis of subclinical cardiac DD in type 2 diabetes.

Recent genome-wide association (GWA) studies have identified new genetic determinants of complex quantitative traits, including plasma triglyceride (TG). We hypothesized that common variants associated with mild TG variation identified in GWA studies would also be associated with severe hypertriglyceridemia (HTG). We studied 132 patients of European ancestry with severe HTG (fasting plasma TG > 10 mmol/l), who had no mutations found by resequencing of candidate genes, and 351 matched normolipidemic controls. We determined genotypes for: GALNT2 rs4846914, TBL2/MLXIPL rs17145738, TRIB1 rs17321515, ANGPTL3 rs12130333, GCKR rs780094, APOA5 rs3135506 (S19W), APOA5 rs662799 (21131T > C), APOE (isoforms) and LPL rs328 (S447X). We found that: (i) genotypes, including those of APOA5 S19W, APOA5 21131T > C, APOE, GCKR, TRIB1 and TBL2/MLXIPL, were significantly associated with severe HTG; (ii) odds ratios for these genetic variables were significant in both univariate and multivariate regression analyses, irrespective of the presence or absence of diabetes or obesity; (iii) a significant fraction—about one-quarter—of the explained variation in disease status was associated with these genotypes. Therefore, common SNPs (single nucleotide polymorphisms) that are associated with mild TG variation in GWA studies of normolipidemic subjects are also associated with severe HTG. Our findings are consistent with the emerging model of a complex genetic trait. At the extremes of a quantitative trait, such as severe HTG, are found the cumulative contributions of both multiple rare alleles with large genetic effects and common alleles with small effects.

The anatomical causes underlying the distribution pattern of abscesses due to the preauricular sinus are presented together with a retrospective study of affected patients. The distribution of the preauricular sinus opening and the abscess position in 27 patients was investigated. To confirm the anatomical factors affecting abscess distribution, auricular and preauricular tissues of cadavers were microscopically and macroscopically examined. Twenty-three of 27 abscesses were concentrated in the preauricular region away from the area where the subcutaneous fat was thick. One was found in the crus helix, and the other three were found in the sinus opening. The preauricular subcutaneous fat layer was contained posteriorly and caudally by the spine of helix, superior auricular ligament, and zygomatic arch. The abscess tended to gravitate toward the preauricular fat layer and it may be misdiagnosed as an infected epidermoid cyst.

Objective— The genetic determinants of severe hypertriglyceridemia (HTG; MIM 144650) in adults are poorly defined. We therefore resequenced 3 candidate genes, namely LPL , APOC2 , and APOA5 , to search for accumulation of missense mutations in patients with severe HTG compared with normolipidemic subjects. Methods and Results— We resequenced >2 million base pairs of genomic DNA from 110 nondiabetic patients with severe HTG and determined the prevalence of coding sequence variants compared with 472 age- and sex-matched normolipidemic controls. We found: (1) heterozygous mutations ( LPL p.Q-12E >11X, p.D25H, p.W86R, p.G188E, p.I194T and p.P207L; APOC2 p.K19T and IVS2–30G>A) in 10.0% of severe HTG patients compared with 0.2% of controls (carrier odds ratio [OR] 52, 95% confidence interval [CI] 8.6 to 319); and (2) an association of the APOA5 p.S19W missense variant with severe HTG (carrier OR 5.5 95% CI 3.3 to 9.1). Furthermore, either rare mutations or the APOA5 p.S19W variant were found in 41.8% of HTG subjects compared with 8.9% of controls (carrier OR 7.4, 95% CI 4.5 to 12.0). Also, heterozygotes for rare mutations had a significantly reduced plasma triglyceride response to fibrate monotherapy. Conclusions— Both common and rare DNA variants in candidate genes were found in a substantial proportion of severe HTG patients. The findings underscore the value of candidate gene resequencing to understand the genetic contribution in complex lipoprotein and metabolic disorders.

To evaluate the potential clinical translation of high-throughput next-generation sequencing (NGS) methods in diagnosis and management of dyslipidemia.Recent NGS experiments indicate that most causative genes for monogenic dyslipidemias are already known. Thus, monogenic dyslipidemias can now be diagnosed using targeted NGS. Targeting of dyslipidemia genes can be achieved by either: designing custom reagents for a dyslipidemia-specific NGS panel; or performing genome-wide NGS and focusing on genes of interest. Advantages of the former approach are lower cost and limited potential to detect incidental pathogenic variants unrelated to dyslipidemia. However, the latter approach is more flexible because masking criteria can be altered as knowledge advances, with no need for re-design of reagents or follow-up sequencing runs. Also, the cost of genome-wide analysis is decreasing and ethical concerns can likely be mitigated. DNA-based diagnosis is already part of the clinical diagnostic algorithms for familial hypercholesterolemia. Furthermore, DNA-based diagnosis is supplanting traditional biochemical methods to diagnose chylomicronemia caused by deficiency of lipoprotein lipase or its co-factors.The increasing availability and decreasing cost of clinical NGS for dyslipidemia means that its potential benefits can now be evaluated on a larger scale.

Background— Inuit are considered to be vulnerable to cardiovascular disease because their lifestyles are becoming more Westernized. During sequence analysis of Inuit individuals at extremes of lipid traits, we identified 2 nonsynonymous variants in low-density lipoprotein receptor ( LDLR ), namely p.G116S and p.R730W. Methods and Results— Genotyping these variants in 3324 Inuit from Alaska, Canada, and Greenland showed they were common, with allele frequencies 10% to 15%. Only p.G116S was associated with dyslipidemia: the increase in LDL cholesterol was 0.54 mmol/L (20.9 mg/dL) per allele ( P =5.6×10 −49 ), which was >3× larger than the largest effect sizes seen with other common variants in other populations. Carriers of p.G116S had a 3.02-fold increased risk of hypercholesterolemia (95% confidence interval, 2.34–3.90; P =1.7×10 −17 ), but did not have classical familial hypercholesterolemia. In vitro, p.G116S showed 60% reduced ligand-binding activity compared with wild-type receptor. In contrast, p.R730W was associated with neither LDL cholesterol level nor altered in vitro activity. Conclusions— LDLR p.G116S is thus unique: a common dysfunctional variant in Inuit whose large effect on LDL cholesterol may have public health implications.

After focal cerebral ischemia, tumor necrosis factor-α deteriorates cerebral edema and survival rate. Therefore, tumor necrosis factor-α neutralization could reduce cerebral microvascular permeability in acute cerebral ischemia. Left middle cerebral artery occlusion for 120 mins followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Antirat tumor necrosis factor-α neutralizing monoclonal antibody with a rat IgG Fc portion (15 mg/kg) was infused intravenously right after reperfusion. Stroke index score, infarct volume, cerebral specific gravity, and the endogenous expression of tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP in the brain tissue were quantified in the ischemic and matched contralateral nonischemic hemisphere. In the antitumor necrosis factor-α neutralizing antibody-treated rats, infarct volume was significantly reduced ( P = 0.014, n = 7; respectively), and cerebral specific gravity was dramatically increased in the cortex and caudate putamen ( P

Objective— Estrogen deficiency is linked with increased low-density lipoprotein (LDL) cholesterol. The hormone receptor mediating this effect is unknown. G-protein estrogen receptor (GPER) is a recently recognized G-protein–coupled receptor that is activated by estrogens. We recently identified a common hypofunctional missense variant of GPER, namely P16L. However, the role of GPER in LDL metabolism is unknown. Therefore, we examined the association of the P16L genotype with plasma LDL cholesterol level. Furthermore, we studied the role of GPER in regulating expression of the LDL receptor and proprotein convertase subtilisin kexin type 9. Approach and Results— Our discovery cohort was a genetically isolated population of Northern European descent, and our validation cohort consisted of normal, healthy women aged 18 to 56 years from London, Ontario. In addition, we examined the effect of GPER on the regulation of proprotein convertase subtilisin kexin type 9 and LDL receptor expression by the treatment with the GPER agonist, G1. In the discovery cohort, GPER P16L genotype was associated with a significant increase in LDL cholesterol (mean±SEM): 3.18±0.05, 3.25±0.08, and 4.25±0.33 mmol/L, respectively, in subjects with CC (homozygous for P16), CT (heterozygotes), and TT (homozygous for L16) genotypes ( P P Conclusions— GPER activation upregulates LDL receptor expression, probably at least, in part, via proprotein convertase subtilisin kexin type 9 downregulation. Furthermore, humans carrying the hypofunctional P16L genetic variant of GPER have increased plasma LDL cholesterol. In aggregate, these data suggest an important role of GPER in the regulation of LDL receptor expression and consequently LDL metabolism.

We retrospectively studied 21 patients who had difficulty tolerating daily or alternating-day statins. Patients received rosuvastatin at a mean frequency of 1.7 doses per week, and a mean dose of 11.7 mg per week. We assessed lipid profiles at baseline and after at least 3 months of therapy. We found that total and low-density lipoprotein cholesterol were reduced by 2.03 ± 2.04 and 1.31 ± 0.83 mmol/L (27.9% and 31.5%), respectively, from baseline (both P < 0.001). Thus, in patients with statin intolerance, infrequent low-dose rosuvastatin significantly improved low-density lipoprotein cholesterol and was well tolerated over the long term.

Introduction: Estrogen deficiency is linked with dyslipidemia, especially in postmenopausal women, through a poorly understood mechanism. GPER is a recently recognized GPCR which is activated by estrogens. However, the role of GPER in mediating estrogen’s effects on lipid metabolism is unknown. We recently identified a common hypofunctional missense variant of GPER, namely P16L (allele frequency ~ 20%). We studied association of this with plasma LDL cholesterol levels. Further, we studied the role of GPER in regulating expression of the LDL receptor. Methods: Our discovery cohort was a genetically isolated population of Northern European descent (n=415), and our validation cohort consisted of 505 normal, healthy subjects 18-56 years of age from London, Ontario. Genomic DNA was extracted from whole blood and genotyped for GPER using a dedicated TaqMan assay. Additionally we examined the role of GPER on the regulation of LDL receptor expression by treatment with the GPER agonist, G1. Results: In the discovery cohort, the GPER P16L genetic variant was associated with a significant gene-dosage related increase in LDL cholesterol (CC [homozygous wild type] =3.18±0.84 (mean+SD); CT [heterozygote] =3.25±0.80; and TT [homozygous variant] =4.25±0.87 mmol/L, p Conclusion: GPER activation upregulates LDL receptor expression. Further, carrying the hypofunctional P16L genetic variant of GPER, increases plasma LDL cholesterol in humans. In aggregate these data suggest an important role of GPER in regulation of LDL receptor expression and consequently LDL metabolism.

2 Abstract Trinidadians of South Asian origin have a high prevalence of cardiovascular disease and diabetes compared to Trinidadians of African ori- gin. The degree to which these differences are related to genetic and/or envi- ronmental factors is unclear. To determine whether there might be a genetic basis for this difference in prevalence of deleterious phenotypes we exam- ined allele frequencies for candidate genes in atherosclerosis and diabetes. We genotyped 81 consecutive neonates of African origin and 103 consecu- tive neonates of South Asian origin. We evaluated common polymorphisms in 11 candidate genes for atherosclerosis and diabetes. We found differences between the two subpopulations in the allele frequencies of several candidate genes, including APOE, LIPC, APOC3, PON1, PON2, and PPP1R3. Howev- er, the differences in the allele frequencies were not all consistent with the pattern of CHD expression between these two ethnic groups in adulthood. Thus, differences in genetic architecture alone may not explain the wide dis- parities in disease prevalence between these two subpopulations. It is very likely that environmental factors, or unmeasured genetic factors, influence the genetic susceptibility to disease in these subpopulations.

Objectives: We hypothesized that common variation in the angiotensinogen (AGT), beta-3-adrenergic receptor, intestinal fatty acid‐ binding protein, serum paraoxonase, paraoxonase-2, hepatic lipase, apolipoprotein E (APOE), and Werner helicase (WRN) genes would be associated with variation in biochemical phenotypes in a previously unstudied neonatal sample. Design and methods: We examined associations of both nongenetic and genetic variables with plasma lipoprotein traits in neonates from Trinidad. Results: Among nongenetic variables, we found significant associations between plasma concentrations of: 1) lipoprotein(a) [Lp(a)] and both ethnicity (p 5 0.037) and birth weight (p 5 0.001); 2) total cholesterol and gender (p 5 0.010); 3) triglyceride and birth weight (p 5 0.035); and 4) apolipoprotein AI and gender (p 5 0.016). Among genetic variables, we found that: 1) common variation on chromosome 1q in AGT codon 235 was significantly associated with variation in plasma apolipoproteins AI (p , 0.0001) and B (p 5 0.013); 2) common variation in WRN at codon 1367 was significantly associated with variation in plasma Lp(a) (p , 0.0001); and 3) common variation in APOE at codons 112 and 158 was significantly associated with variation in plasma triglycerides (p 5 0.013). Conclusions: The associations with AGT and WRN are novel and may have resulted either from a direct influence of the genetic variants or through linkage disequilibrium with other functional loci, such as the familial combined hyperlipidemia locus on chromosome 1q in the case of AGT. Despite the fact that there are some limitations in making determinations from cord blood, the results suggest that there may be genetic determinants of plasma lipoproteins in neonates. Copyright © 1999 The Canadian Society of Clinical Chemists