Samantha L. Rosenthal

A population-specific missense variant, rs373863828 (G>A, p.Arg457Gln), has been identified in Pacific Islanders and is associated with increased body mass index and lower odds of type 2 diabetes. However, the mechanism underlying these associations is unknown. The variant is located in exon 5 of CREBRF , a gene which is rapidly upregulated in response to nutrient deprivation. This study aimed to assess the effect of rs373863828 on gene expression in adipose tissue from Samoan adults from the Soifua Manuia (“Good Health”) study. Needle subcutaneous adipose (upper buttock) biopsies were obtained in Apia, Samoa, from fasting Samoans without diabetes. RNA isolated from adipose tissue (n=91, 65.9% female) was subjected to bulk paired-end mRNA-sequencing (2×150 base pair). Differential gene expression among the three rs373863828 genotypes (GG, AG, AA) was assessed using a likelihood-ratio test implemented in DESeq2, and overrepresentation analysis for significant differentially expressed genes (DEGs) ( p _adj < 0.1) was performed with clusterProfiler and oRG.DB. Bulk expression was deconvoluted to determine the cell type composition of the adipose tissue. Twenty-two DEGs were associated with rs373863828 genotype including CBX7 ( p _adj =0.026) and SELENOM ( p _adj = 0.004), both of which have roles in adipogenesis and metabolism. A single expression profile comprising 13 of the 22 DEGs corresponding to CREBRF genotype was identified, and overrepresentation analysis supported this finding, with terms such as “keratinocyte differentiation” and “skin development” enriched among significant DEGs. However, PERMANOVA of adipose cell type proportions showed no difference ( p > 0.05) among the three genotypes. rs373863828 genotype was associated with expression of 22 genes in whole subcutaneous adipose tissue of this sample of fasting Samoan adults without diabetes. Future work should assess cell- and tissue-specific rs373863828 genotype effects using a combination of single-cell approaches and human cell models with perturbations associated with nutritional stress. AUTHOR SUMMARY: A genetic variant (change in DNA, G >A) is associated with higher body mass index and lower risk for type 2 diabetes. This variant, called rs373863828, is only present in people of Pacific Islander ancestry, who are underrepresented in genetic and genomic studies, and is in the CREBRF gene, which is not well-understood. Studies in mice, flies, and cells from humans and mice suggest CREBRF is important for cellular growth and stress response, and that rs373863828 increases these functions. In this study, we tested the hypothesis that CREBRF affects these cellular processes by controlling gene expression—how genes are “turned on or off” –in fat tissue from Samoan adults as part of the Soifua Manuia (“Good Health”) study. We found 22 genes that were affected by how many G to A changes (0, 1, or 2) an individual has at this position in CREBRF . We also found that the variant did not affect the proportion of the cell types that make up adipose tissue. This work helps us to better understand the function of the CREBRF gene in human fat and gives some clues about how the variant affects BMI and type 2 diabetes risk in healthy Samoan adults.

Purpose: The aim of this study was to evaluate the color stability of 3D printed resin disks using spectral reflectance data obtained at different time periods after immersion in various staining solutions. The color stability of 3D-printed temporary crowns is clinically important, as it directly affects the esthetic outcome and patient satisfaction during the provisional phase of treatment. Materials and methods: Forty identical round disk specimens measuring 10 mm in diameter and 2 mm in thickness were fabricated using CAD/CAM 3D printing resin (shade B1). Half of the specimens (n = 20) were polished using an acrylic bur and medium pumice. The remaining specimens were unpolished (n = 20). Each group of disks was then immersed in one of the following immersion solutions: artificial saliva, black tea, carrot juice, and red wine. Color difference ΔE was evaluated using the spectrophotometer, a spectral reflectance instrument, at baseline, day 1, week 1, week 2 and week 3, against a white background. Comparisons between polished and unpolished disks at each time point were conducted using Mann–Whitney tests. Differences among the staining solutions at each time point for both polished and unpolished disks were analyzed using a one-way ANOVA with Tukey’s post hoc test. Results: Color difference ΔE was measured using the CIELAB formula. The mean ΔE values of each group were calculated. The greatest difference in color was observed in the unpolished and polished disks immersed in red wine. Polished disks showed less color difference when compared to unpolished disks. Significant differences in ΔE were detected between polished and unpolished disks immersed in red wine at week 1 (p = 0.0159), week 2 (p = 0.0079) and week 3 (p = 0.0079) and in carrot juice at week 3 (p = 0.0317). Conclusions: Immersion of 3D printed disks in different staining solutions caused detectable color difference in the tested materials, which was relative to the immersion duration and the staining solution used. The color of the 3D printed resins is influenced by the surface finishing, which may result in visually perceptible color differences. The color stability of 3D printied materials should be improved to provide long-term esthetics.

Background: The prevalence of obesity and diabetes in Samoa, like many other Pacific Island nations, has reached epidemic proportions. Although the etiology of these conditions can be largely attributed to the rapidly changing economic and nutritional environment, a recently identified genetic variant, rs373863828 (CREB 3 regulatory factor, CREBRF: c.1370G>A p.[R457Q]) is associated with increased odds of obesity, but paradoxically, decreased odds of diabetes. Objective: The overarching goal of the Soifua Manuia (Good Health) study was to precisely characterize the association of the CREBRF variant with metabolic (body composition and glucose homeostasis) and behavioral traits (dietary intake, physical activity, sleep, and weight control behaviors) that influence energy homeostasis in 500 adults. Methods: A cohort of adult Samoans who participated in a genome-wide association study of adiposity in Samoa in 2010 was followed up, based on the presence or absence of the CREBRF variant, between August 2017 and March 2019. Over a period of 7-10 days, each participant completed the main study protocol, which consisted of anthropometric measurements (weight, height, circumferences, and skinfolds), body composition assessment (bioelectrical impedance and dual-energy x-ray absorptiometry), point-of-care glycated hemoglobin measurement, a fasting blood draw and oral glucose tolerance test, urine collection, blood pressure measurement, hand grip strength measurement, objective physical activity and sleep apnea monitoring, and questionnaire measures (eg, health interview, cigarette and alcohol use, food frequency questionnaire, socioeconomic position, stress, social support, food and water insecurity, sleep, body image, and dietary preferences). In January 2019, a subsample of the study participants (n=118) completed a buttock fat biopsy procedure to collect subcutaneous adipose tissue samples. Results: Enrollment of 519 participants was completed in March 2019. Data analyses are ongoing, with results expected in 2020 and 2021. Conclusions: While the genetic variant rs373863828, in CREBRF, has the largest known effect size of any identified common obesity gene, very little is currently understood about the mechanisms by which it confers increased odds of obesity but paradoxically lowered odds of type 2 diabetes. The results of this study will provide insights into how the gene functions on a whole-body level, which could provide novel targets to prevent or treat obesity, diabetes, and associated metabolic disorders. This study represents the human arm of a comprehensive and integrated approach involving humans as well as preclinical models that will provide novel insights into metabolic disease. International Registered Report Identifier (IRRID): RR1-10.2196/17329

Objectives: Studies have demonstrated that rs373863828, a missense variant in CREBRF, is associated with a number of anthropometric traits including body mass index (BMI), obesity, percent body fat, hip circumference, and abdominal circumference. Given the biological relationship between height and adiposity, we hypothesized that the effect of this variant on BMI might be due in part to an association of this variant with height.Methods: We tested the hypothesis that minor allele of rs373863828 is associated with height in a Samoan population in two adult cohorts and in a separate cohort of children (age 5‐18 years old) using linear mixed modeling.Results: We found evidence of a strong relationship between rs373863828 and greater mean height in Samoan adults (0.77 cm greater average height for each copy of the minor allele) with the same direction of effect in Samoan children.Conclusions: These results suggest that the missense variant rs373863828 in CREBRF, first identified through an association with larger BMI, may be related to an underlying biological mechanism affecting overall body size including stature.

Coronary artery disease (CAD) is a complex disorder involving both genetic and non-genetic factors. Genome-wide association studies (GWAS) have identified hundreds of single nucleotides polymorphisms (SNPs) tagging over > 40 CAD risk loci. We hypothesized that some non-coding variants might directly regulate the gene expression rather than tagging a nearby locus. We used RegulomeDB to examine regulatory functions of 58 SNPs identified in two GWAS and those SNPs in linkage disequilibrium (LD) (r² ≥ 0.80) with the GWAS SNPs. Of the tested 1200 SNPs, 858 returned scores of 1–6 by RegulomeDB. Of these 858 SNPs, 97 were predicted to have regulatory functions with RegulomeDB score of < 3. Notably, only 8 of the 97 predicted regulatory variants were genome-wide significant SNPs (LIPA/rs2246833, RegulomeDB score = 1b; ZC3HC1/rs11556924, CYP17A1-CNNM2-NT5C2/rs12413409, APOE-APOC1/rs2075650 and UBE2Z/rs46522, each with a RegulomeDB score = 1f; ZNF259-APOA5-APOA1/rs964184, SMG6/rs2281727 and COL4A1-COL4A2/rs4773144, each with a RegulomeDB score = 2b). The remainder 89 functional SNPs were in linkage disequilibrium with GWAS SNPs. This study supports the hypothesis that some of the non-coding variants are true signals via regulation of gene expression at transcription level. Our study indicates that RegulomeDB is a useful database to examine the putative functions of large number of genetic variants and it may help to identify a true association among multiple tagged SNPs in a complex disease, such as CAD.

Late-onset Alzheimer's disease (LOAD) is a multifactorial disorder with over twenty loci associated with disease risk. Given the number of genome-wide significant variants that fall outside of coding regions, it is possible that some of these variants alter some function of gene expression rather than tagging coding variants that alter protein structure and/or function. RegulomeDB is a database that annotates regulatory functions of genetic variants. In this study, we utilized RegulomeDB to investigate potential regulatory functions of lead single nucleotide polymorphisms (SNPs) identified in five genome-wide association studies (GWAS) of risk and age-at onset (AAO) of LOAD, as well as SNPs in LD (r²≥0.80) with the lead GWAS SNPs. Of a total 614 SNPs examined, 394 returned RegulomeDB scores of 1–6. Of those 394 variants, 34 showed strong evidence of regulatory function (RegulomeDB score ZCWPW1/rs1476679, CLU/rs1532278 and ABCA7/rs3764650). This study further supports the assumption that some of the non-coding GWAS SNPs are true associations rather than tagged associations and demonstrates the application of RegulomeDB to GWAS data.