Using genetics to test the causal relationship of total adiposity and periodontitis: Mendelian randomization analyses in the Gene-Lifestyle Interactions and Dental Endpoints (GLIDE) Consortium

Background: The observational relationship between obesity and periodontitis is widely known, yet causal evidence is lacking. Our objective was to investigate causal associations between periodontitis and body mass index (BMI).Methods: We performed Mendelian randomization analyses with BMI-associated loci combined in a genetic risk score (GRS) as the instrument for BMI. All analyses were conducted within the Gene-Lifestyle Interactions and Dental Endpoints (GLIDE) Consortium in 13 studies from Europe and the USA, including 49 066 participants with clinically assessed (seven studies, 42.1% of participants) and self-reported (six studies, 57.9% of participants) periodontitis and genotype data (17 672/31 394 with/without periodontitis); 68 761 participants with BMI and genotype data; and 57 871 participants (18 881/38 990 with/without periodontitis) with data on BMI and periodontitis.Results: In the observational meta-analysis of all participants, the pooled crude observational odds ratio (OR) for periodontitis was 1.13 [95% confidence interval (CI): 1.03, 1.24] per standard deviation increase of BMI. Controlling for potential confounders attenuated this estimate (OR = 1.08; 95% CI:1.03, 1.12). For clinically assessed periodontitis, corresponding ORs were 1.25 (95% CI: 1.10, 1.42) and 1.13 (95% CI: 1.10, 1.17), respectively. In the genetic association meta-analysis, the OR for periodontitis was 1.01 (95% CI: 0.99, 1.03) per GRS unit (per one effect allele) in all participants and 1.00 (95% CI: 0.97, 1.03) in participants with clinically assessed periodontitis. The instrumental variable meta-analysis of all participants yielded an OR of 1.05 (95% CI: 0.80, 1.38) per BMI standard deviation, and 0.90 (95% CI: 0.56, 1.46) in participants with clinical data.Conclusions: Our study does not support total adiposity as a causal risk factor for periodontitis, as the point estimate is very close to the null in the causal inference analysis, with wide confidence intervals

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Characterization of flavin binding in oxygen‐independent fluorescent reporters

Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen-free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn-on biosensor for vitamin B2

Characterization of flavin binding in oxygen‐independent fluorescent reporters

Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen-free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn-on biosensor for vitamin B2

Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters

<div><p>Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)–namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from <i>Pseudomonas putida</i>, <i>Bacillus subtilis</i>, and <i>Arabidopsis thaliana,</i> using <i>in vitro</i> studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using <i>in vivo</i> studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in <i>Escherichia coli</i>. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4–11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from <i>Arabidopsis thaliana</i> (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in <i>Escherichia coli</i>. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10–40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.</p></div

SDS PAGE analysis of purified FbFPs.

<p>FbFPs were purified using nickel affinity and anion exchange chromatography. A) Purified FbFPs were loaded on a denaturing polyacrylamide gel and allowed to migrate at 160 V for 40 min. Lane 1: iLOV after nickel affinity chromatography, lane 2: iLOV after anion exchange chromatography, lane 3: PpFbFP after nickel affinity chromatography, lane 4: PpFbFP after anion exchange chromatography, lane 5: EcFbFP after nickel affinity chromatography, lane 6: EcFbFP after anion exchange chromatography. B) Purified PpFbFP and iLOV emit cyan-green fluorescence upon excitation using a UV transilluminator.</p

Quantum yield and brightness of FbFPs.

<p>Brightness is quantified as the product of quantum yield and molar extinction coefficient of the fluorophore (flavin mononucleotide). Brightness values are reported for monomeric units of PpFbFP and EcFbFP, which exist as functional dimers.</p