Genetic and seasonal determinants of vitamin D status in Confederated Salish and Kootenai Tribes (CSKT) participants

Background: Vitamin D is a hormone produced in the skin upon ultraviolet B (UVB) radiation. Vitamin D is a crucial regulator of calcium and phosphate levels for bone mineralization and other physiological roles. Vitamin D levels vary globally in human populations due to genetics, geography, and other demographic factors. It is estimated that 20-85 % of the variability in vitamin D levels is driven by genetic variation. To improve our understanding of contributors to vitamin D levels, we conducted a candidate-gene study in partnership with the Confederated Salish and Kootenai Tribes (CSKT). Methods: We recruited 472 CSKT study participants on the Flathead Reservation in Montana. Demographic factors included age, BMI, and gender (185 male and 287 female; ≥ 18 years old). Genomic DNA and plasma were isolated from whole blood. We sequenced 14 vitamin D regulatory candidate genes: CASR, CUBN, CYP2R1, CYP3A4,CYP24A1, CYP27B1, DHCR7, GC, RXRA, RXRB, RXRG, SULT2A1, UGT1A4, and VDR. We also measured plasma levels of vitamin D and vitamin D metabolites by liquid chromatography/mass-spectrometry (LC/MS), including the clinical marker of vitamin D status, 25-hydroxyvitamin D3 [25(OH)D3]. We tested demographic factors as well as common and rare genetic variants for statistical associations with vitamin D levels using bioinformatics software and R statistical programming language code. Results: We identified 7,370 total genetic variants with 8% (n = 585) of them being novel. We identified 60 genetic variants that may be of clinical significance (disease associated or predicted to influence medication response). Vitamin D levels were below sufficiency [25(OH)D3 + 25(OH)D2 levels \u3c 20 ng/mL] in 56 % of CSKT participants across the year. We observed seasonal vitamin D and metabolite level fluctuations in a seasonal, sinusoidal statistical model with peak concentrations in June – August and trough concentrations in December – February. In linear regression analysis, we found that age, BMI, season, and 5 variants in CUBN and CYP3A4 were significantly associated with 25(OH)D3 concentration (p-value\u3c 0.05). In logistic regression, we found that 4 variants in CUBN, CYP3A4, and UGT1A4 were associated with 25(OH)D sufficiency status [25(OH)D3 + 25(OH)D2 levels of 20 ng/mL] (p-value\u3c 0.05). Multivariate linear regression analysis revealed that genetic variation alone explained ~13% of the variability in 25(OH)D3 concentration in CSKT participants. Genetic variation and environmental factors together explained ~23 % of the variability in 25(OH)D3 concentration in CSKT participants. It is likely that genetic variation in additional genes and other environmental factors (e.g., dietary vitamin D intake) that were not included in this study explain the remaining variability in 25(OH)D3 concentration. Conclusion: This research addresses the need for increased inclusion of American Indian and Alaska Natives in precision medicine health research. We are the first to describe the contribution of season and genetics to vitamin D levels in an American Indian population. Our next steps will be to use these findings to perform mechanistic studies and develop interventional strategies for the CSKT people

Genetic and seasonal contributions to variability in vitamin D levels among American Indians

Purpose: Vitamin D is an essential hormone in maintenance of calcium and phosphate homeostasis for adequate bone mineralization with roles in the immune system as well. Sources of vitamin D include natural synthesis in the skin upon ultraviolet B (UVB) radiation, dietary intake from plant and animal sources, and supplementation. Vitamin D levels, measured by the primary circulating vitamin D metabolite, 25-hydroxyvitamin D (25(OH)D), are variable across individuals due to latitude, season, diet, gender, disease states, medication use, and genetics. Populations living at northern latitudes (~400N) such as American Indians of the Confederated Salish and Kootenai Tribes (CSKT) are at increased susceptibility of having lower vitamin D levels in the winter months because there is decreased UVB exposure. There are currently no published data that exist on the genetic and seasonal influence on vitamin D status in CSKT people. Methods: We recruited 472 research participants from the Confederated Salish and Kootenai Tribes (CSKT) (185 male and 287 female; 18 years or older) at various sites in the Flathead Reservation in western Montana. Demographic factors were collected (e.g., age, body mass index, and gender). Genomic DNA and plasma were isolated from whole blood. We sequenced 12 candidate genes: 7-dehydrocholesterol reductase (DHCR7), calcium-sensing receptor (CASR), cubulin (CUBN), cytochrome P450 enzymes (CYP2R1, CYP27B1, CYP24A1, and CYP3A4), retinoid X receptors (RXR alpha, beta, and gamma), sulfotransferase family 2A member 1 (SULT2A1), UDP-glucuronosyltransferase family 1A3 and 1A4 (UGT1A3 and UGT1A4), vitamin D binding protein (GC), and vitamin D receptor (VDR). We also measured circulating levels of vitamin D and metabolites: vitamin D3 and vitamin D2, 25-hydroxyvitamin D [25(OH)D3 and 25(OH)D2], 1,25-dihydroxyvitamin D3 and D2 [1,25(OH)2D3 and 1,25(OH)2D2], 24R,25-dihydroxyvitamin D3 [24R,25(OH)D3], and 4-b,25-dihydroxyvitamin D3 [4b,25(OH)2D3]. Candidate genes were resequenced with Illumina next generation sequencing technology and vitamin D and metabolites were quantitated with liquid chromatography mass spectrometry (LC-MS/MS). The data we collected was summarized using a pipeline state of the art bioinformatics software. Originality: This research addresses the need for increased inclusion of American Indian and Alaska Natives in precision medicine health research. We have characterized genetic variation in vitamin D endocrinology genes and seasonal variability in vitamin D and vitamin D metabolite levels for the first time in CSKT people. Significance: We observed known and novel genetic variation in the CSKT population based on the presence or absence of genetic variants in the National Center for Biotechnology Information (NCBI) Single Nucleotide Polymorphism Database (dbSNP). We observed clinically relevant genetic variants that are associated with disease and variability in response to medication based on presence in the NCBI ClinVar and Pharmacogenomics Knowledge Base (PharmGKB) databases. Vitamin D and metabolite levels varied in a seasonal, fluctuating way, as expected, with lowest levels in January, February, and March months and highest levels in June, July, and August months. A significant percentage of CSKT participants had measured vitamin D levels below sufficiency (~43%). We will use these data to design an interventional strategy to address the low vitamin D levels we observed in CSKT people

Cytochrome P450 Genetic Variation Associated with Tamoxifen Biotransformation in American Indian and Alaska Native People

Despite evidence that pharmacogenetics can improve tamoxifen pharmacotherapy, there are few studies with American Indian and Alaska Native (AIAN) people. We examined variation in cytochrome P450 (CYP) genes (CYP2D6, CYP3A4, CYP3A5, and CYP2C9) and tamoxifen biotransformation in AIAN patients with breast cancer (n = 42) from the Southcentral Foundation in Alaska and the Confederated Salish and Kootenai Tribes in Montana. We tested for associations between CYP diplotypes and plasma concentrations of tamoxifen and metabolites. Only the CYP2D6 variation was significantly associated with concentrations of endoxifen (P = 0.0008) and 4-hydroxytamoxifen (P = 0.0074), tamoxifen's principal active metabolites, as well as key metabolic ratios. The CYP2D6 was also the most significant predictor of active metabolites and metabolic ratios in a multivariate regression model, including all four genes as predictors, with minor roles for other CYP genes. In AIAN populations, CYP2D6 is the largest contributor to tamoxifen bioactivation, illustrating the importance of validating pharmacogenetic testing for therapy optimization in an understudied population