Association between genetic variants in key vitamin-D-pathway genes and external apical root resorption linked to orthodontic treatment.

This study evaluated the association between single-nucleotide polymorphisms (SNPs) in vitamin-D-related genes and the amount of external apical root resorption linked to orthodontic treatment. One hundred and forty-three individuals were assessed. The amount of external apical root resorption of upper central incisors (EARRinc ) and lower first molars (EARRmol ) were evaluated in radiographs. Seven SNPs were genotyped across four genes including the vitamin D receptor [VDR], group-specific component [GC], cytochrome P450 family 27 subfamily B member 1 [CYP27B1], and cytochrome P450 family 24 subfamily A member 1 [CYP24A1]. Linear regressions were implemented to determine allele-effects on external apical root resorption. Individuals carrying the AA genotype in VDR rs2228570 had a 21% higher EARRmol than those having AG and GG genotypes (95% CI: 1.03,1.40). EARRmol in heterozygous rs2228570, was 12% lower than for homozygotes (95%CI: 0.78,0.99). Participants with the CCG haplotype (rs1544410-rs7975232-rs731236) in VDR had an EARRmol 16% lower than those who did not carry this haplotype. Regarding CYP27B1 rs4646536, EARRinc in participants who had at least one G allele was 42% lower than for homozygotes AA (95%CI: 0.37,0.93). Although these results did not remain significant after multiple testing adjustment, potential associations may still be suggested. Further replication studies are needed to confirm or refute these findings.

Vitamin D metabolic pathway genes polymorphisms and vitamin D levels in association with neonatal hyperbilirubinemia in China: a single-center retrospective cohort study.

BACKGROUND: Neonatal hyperbilirubinemia (NH) is a major cause of hospitalization after birth. Previous studies indicated that vitamin D deficiency might play an important role in NH susceptibility, but the results were controversial. Meanwhile, there has been limited description of the association between vitamin D related genes single nucleotide polymorphisms (SNP) and NH susceptibility. We aimed to investigate the vitamin D metabolic pathway genes polymorphisms and vitamin D levels with NH susceptibility. METHODS: We retrospectively analyzed the clinical data, vitamin D levels and its metabolic pathway gene polymorphisms of 187 NH neonates and 149 controls at Tianjin Children's Hospital/Tianjin University Children's Hospital between April 2019 and August 2022. Vitamin D levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and the genetic polymorphism of NADSYN1/DHCR7, GC, CYP2R1, CYP24A1 and CYP27B1 was detected by high resolution melting (HRM) analysis. RESULTS: The frequency of vitamin D deficiency (25(OH)D < 15 ng/mL) was significantly increased in the NH group compared to controls. TT genotype of rs12785878 and GT genotype of rs10877012 were protective factors of vitamin D deficiency and NH, and GT genotype and dominant model carriers of rs12785878 had a higher risk of severe NH than the GG genotype carriers (GT genotype: OR: 2.43; 95% CI: 1.22-4.86; P = 0.012, dominant model: OR: 1.97; 95% CI: 1.04-3.73; P = 0.037). GC gene haplotype was associated with vitamin D deficiency. No significant SNP-SNP and SNP-vitamin D levels interaction combinations were found. CONCLUSIONS: There were associations among NH, vitamin D deficiency and NADSYN1/DHCR7 and CYP27B1 polymorphisms, TT genotype of rs12785878 and GT genotype of rs10877012 could reduce the risk of vitamin D deficiency and NH. Furthermore, rs12785878 was significantly associated with severe NH.

Alterations of subset and cytokine profile of peripheral T helper cells in PBMCs from Multiple Sclerosis patients or from individuals with MS risk SNPs near genes CYP27B1 and CYP24A1.

T helper cells play an important role in the aetiology of Multiple Sclerosis (MS). Vitamin D has an anti-inflammatory effect on T helper cells and can affect onset and pathogenesis of MS. Two genes of the metabolic Vitamin D pathway expressed by activated T helper (Th) cells have been identified as MS risk genes by genome-wide association studies, CYP27B1 (25(OH)D3 1-alpha-hydroxylase) and CYP24A1 (1,25(OH)2D3 24-alpha-hydroxylase). Therefore, we hypothesize that the MS risk alleles around gene CYP27B1 and CYP24A1 are associated with the altered inflammatory profile of peripheral Th cells in PBMCs both ex vivo and in vitro potentially influencing the pathogenesis of MS. PBMCs from MS patients (41 RRMS patients in their remitting stage and 4 SPMS patients) and 12 healthy controls were collected, subpopulation of Th cells in PBMCs and cytokine profile were tested by Flow cytometry and Cytometric Bead Array (CBA), respectively. MS risk SNPs were genotyped by allele-specific PCR analysis. Data were analysed using nonparametric tests and linear regression for adjusting multiple factors. The proportion of Th17.1, Th17 and Th1 cells were all associated with MS while the proportions of Th2 (significant) and Th17 (near significant) cells were correlated with the expanded disability scale score of MS patients. Additionally, we found a MS-specific dysregulation in the IL-6 and TNF production of Th cells in Concanavalin A-stimulated PBMCs. Furthermore, the risk allele rs2248359-C (near gene CYP24A1) showed a consistent inhibitory effect on the proportions of Th1 and Th17.1 cells, and the presence of the homozygous risk allele rs703842-AA (near gene CYP27B1) reduced the production of IL-2. In conclusion, both MS disease and its risk alleles near Vitamin D metabolism genes influence the inflammatory profile of T helper cells in PBMCs.

Abnormal pattern of vitamin D receptor-associated genes and lncRNAs in patients with bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a multifactorial condition. Several signaling pathways affect development of this disorder. With the purpose of exploring the role of vitamin D receptor (VDR) signaling in this disorder, we measured expression of selected mRNA coding genes and long non-coding RNAs (lncRNAs) in this pathway in patients versus normal subjects. METHODS: We measured expression of VDR-associated lncRNAs and mRNAs (SNHG6, MALAT1, Linc00511, Linc00346, VDR and CYP27B1) in the peripheral blood of BD patients vs. healthy individuals. RESULTS: Expression of SNHG6 was significantly higher in cases vs. controls (Posterior beta = 1.29, P value < 0.0001. Subgroup analysis by sex revealed significant results in both subgroups (P value < 0.0001 and P value = 0.023 for males and females, respectively). Expression of CYP27B1 was up-regulated in cases vs. controls (Posterior beta = 0.415, P < 0.0001). Such pattern was also detected among males (P < 0.0001), but not females (P = 0.419). Similarly, MALAT1 and Linc00346 were up-regulated in total cases vs. controls (Posterior beta = 0.694, P < 0.0001 and Posterior beta = 0.4, P = 0.012, respectively) and in male cases compared with male controls (Posterior beta = 0.712, P < 0.0001 and Posterior beta = 0.41, P value = 0.038, respectively). Expression of VDR was up-regulated in total cases compared with controls (Posterior beta = 0.683, P value = 0.001). Finally, expression of Linc00511 was not different between groups. MALAT1, SNHG6, CYP27B1, VDR and Linc00346 had AUC values of 0.95, 0.94, 0.91, 0.85 and 0.83 in differentiation of male patients from controls, respectively. CONCLUSION: The current study suggests VDR-associated genes as possible markers for BD.

Vitamin D3 Metabolic Enzymes in the Porcine Uterus: Expression, Localization and Autoregulation by 1,25(OH)2D3 In Vitro.

The role of vitamin D3 has been confirmed in female reproductive organs. This study aimed to examine vitamin D3 metabolic enzymes, i.e., CYP27B1 and CYP24A1, mRNA transcript and protein abundance, and protein localization in the uterus of pigs on days 2-5, 10-12, 15-16 and 18-20 of the estrous cycle. Additionally, we determined 1,25(OH)2D3 concentration in uterine flushings and the effect of 1,25(OH)2D3 (10, 50 and 100 ng/mL) in vitro on CYP27B1 and CYP24A1 mRNA transcript abundance in endometrial and myometrial slices. In the endometrium, a greater CYP27B1 mRNA transcript abundance was noted on days 10-12 and 18-20 than on days 15-16, whereas encoded protein abundance was greater on days 18-20 when compared to days 15-16. Endometrial CYP24A1 mRNA transcript abundance was greater on days 18-20 than on days 10-12 and 15-16. In the myometrium, CYP27B1 mRNA transcript abundance was greater on days 18-20 than on days 2-5 and 15-16, while protein abundance was larger in slices collected on days 18-20 than on days 15-16. Neither CYP24A1 mRNA transcript nor encoded protein abundance were detected in the myometrium. The highest 1,25(OH)2D3 concentration in uterine flushings was observed on days 18-20. Furthermore, the 1,25(OH)2D3 increased the abundance of the CYP24A1 mRNA transcript in endometrial slices. Overall, our results suggest that porcine uterus is an extra-renal site of vitamin D3 metabolism. Both the endometrium and the myometrium possess the ability to synthesize vitamin D3, while only the endometrium contributes to its catabolism.

Metabolism of 25-Hydroxy-Vitamin D in Human Macrophages Is Highly Dependent on Macrophage Polarization.

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages.

The role of vitamin D-synthesizing enzyme CYP27B1 in systemic lupus erythematosus.

BACKGROUND: To measure the expression of 1α-hydroxylase (CYP27B1) and serum 25(OH)D concentration in systemic lupus erythematosus (SLE) and to investigate the role of CYP27B1 in SLE. METHODS: Seventy-seven SLE patients and 35 healthy controls (HCs) were enrolled from September 2017 to January 2020. The study design is cross-sectional. mRNA expression of CYP27B1 in peripheral blood mononuclear cells (PBMCs) was measured by reverse-transcription quantitative PCR, the protein level of CYP27B1 was quantified by western blotting, and the serum level of 25(OH) D was determined by an enzyme-linked immunosorbent assay. RESULTS: The mRNA expression of CYP27B1 in PBMCs was significantly lower in SLE patients than in HCs (p < 0.001), and the protein quantification confirmed that CYP27B1 expression was lower in SLE patients than in HCs (p = 0.001). Among SLE patients, the prevalence of lupus nephritis was higher in a subgroup with lower CYP27B1 mRNA expression than in a subgroup with normal CYP27B1 mRNA expression (41.07% vs. 14.28%, p = 0.028). The mRNA expression of CYP27B1 negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (r = -0.331, p = 0.003). Serum 25(OH)D concentration was lower in SLE patients than in HCs (37.64 ± 19.89 vs. 50.58 ± 12.74 ng/mL, mean ± SD, p = 0.003). DISCUSSION: The expression of CYP27B1 in PBMCs may be related to SLE pathogenesis, disease activity, and nephritis.

Does Genotype-Phenotype Correlation Exist in Vitamin D-Dependent Rickets Type IA: Report of 13 New Cases and Review of the Literature.

Vitamin D-dependent rickets type IA (VDDR-IA) is caused by biallelic mutations in CYP27B1. Data regarding genotype-phenotype correlation in VDDR-IA are scarce. Here, we aimed to investigate clinical/genotypic features and long-term follow-up of 13 new cases with VDDR-IA and genotype-phenotype correlation of reported cases in the literature. Thirteen patients with VDDR-IA were evaluated. Eight patients had reached their final height at the time of the study and, for whom, long-term outcome data were analyzed. Further, all VDDR-IA patients in the literature (n:183) were analyzed and clinical-genetic features were recorded. The median age of diagnosis was 2.55 ± 1.13 (1.0-12) years. Initial diagnoses before referral to our clinic were nutritional rickets (n:7), hypophosphatemic rickets (n:2), and pseudohypoparathyroidism (n:1). All had biochemical evidence suggestive of VDDR-IA; except one with elevated 1,25(OH)2D3 and another with hyperphosphatemia, in whom pseudohypoparathyroidism was excluded with molecular tests. Combined analyses of our cohort and other series in the literature demonstrated that three most common CYP27B1 mutations are p.F443Pfs*24, c.195 + 2T > G, and p.V88Wfs*71. In Turkish population, p.K192E mutation along with the former two is the most common mutations. Comparison of clinical features demonstrated that c.195 + 2T > G mutation causes the most severe and p.K192E mutation causes the least severe phenotype with respect to age and height at presentation and calcitriol requirement. We found a clear genotype-phenotype correlation in VDDR-IA, notably CYP27B1 intronic c.195 + 2T > G mutation causes a more severe phenotype with lower height SDS at presentation and, higher calcitriol requirement, while less severe phenotype occurs in p.K192E mutation.

Evaluation of expression of VDR-associated lncRNAs in COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been shown to cause serious health problems among them is the Acute Respiratory Distress syndrome (ARDS). Vitamin D receptor (VDR) signaling possibly partakes in the pathophysiology of this devastating complication. METHODS: In the current project, we have appraised expression levels of VDR, CYP27B1 and a number of associated lncRNAs in the circulation of COVID-19 patients versus healthy subjects using real-time PCR method. RESULTS: Expression of SNHG6 was considerably lower in COVID-19 patients compared with control subjects (Ratio of mean expression (RME) = 0.22, P value = 7.04E-05) and in both female and male COVID-19 patients compared with sex-matched unaffected individuals (RME = 0.32, P value = 0.04 and RME = 0.16, P value = 0.000679683, respectively). However, its expression was similar among ICU-hospitalized and non-ICU patients. Similarly, expression of SNHG16 was lower in in COVID-19 patients compared with controls (RME = 0.20, P value = 5.94E-05) and in both female and male patients compared with sex-matched controls (RME = 0.32, P value = 0.04 and RME = 0.14, P value = 0.000496435, respectively) with no significant difference among ICU-hospitalized and non-ICU hospitalized patients. Expression of VDR was lower in COVID-19 patients compared with controls (RME = 0.42, P value = 0.04) and in male patients compared with male controls (RME = 0.27, P value = 0.02). Yet, expression of VDR was statistically similar between female subgroups and between ICU-hospitalized and non-ICU hospitalized patients. Expression levels CYP27B, Linc00511 and Linc00346 were similar among COVID-19 patients and healthy subjects or between their subgroups. Significant correlations have been detected between expression levels of VDR, CYP27B and SNHG6, SNHG16, Linc00511 and Linc00346 lncRNAs both among COVID-19 patients and among healthy controls with the most significant ones being SNHG6 and SNHG16 (r = 0.74, P value = 3.26e-17 and r = 0.81, P = 1.54e-22, respectively). CONCLUSION: Combination of transcript levels of VDR, CYP27B and SNHG6, SNHG16, Linc00511 and Linc00346 could differentiate patients from controls with AUC = 0.76, sensitivity = 0.62 and specificity = 0.81. The current data potentiate SNHG6, SNHG16 and VDR as possible contributors in COVID-19 infection but not in the severity of ARDS.

Vitamin D Metabolism Is Dysregulated in Asthma and Chronic Obstructive Pulmonary Disease.

Rationale: Vitamin D deficiency is common in patients with asthma and chronic obstructive pulmonary disease (COPD). Low 25-hydroxyvitamin D (25[OH]D) levels may represent a cause or a consequence of these conditions.Objectives: To determine whether vitamin D metabolism is altered in asthma or COPD.Methods: We conducted a longitudinal study in 186 adults to determine whether the 25(OH)D response to six oral doses of 3 mg vitamin D3, administered over 1 year, differed between those with asthma or COPD versus control subjects. Serum concentrations of vitamin D3, 25(OH)D3, and 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) were determined presupplementation and postsupplementation in 93 adults with asthma, COPD, or neither condition, and metabolite-to-parent compound molar ratios were compared between groups to estimate hydroxylase activity. Additionally, we analyzed 14 datasets to compare expression of 1α,25(OH)2D3-inducible gene expression signatures in clinical samples taken from adults with asthma or COPD versus control subjects.Measurements and Main Results: The mean postsupplementation 25(OH)D increase in participants with asthma (20.9 nmol/L) and COPD (21.5 nmol/L) was lower than in control subjects (39.8 nmol/L; P = 0.001). Compared with control subjects, patients with asthma and COPD had lower molar ratios of 25(OH)D3-to-vitamin D3 and higher molar ratios of 1α,25(OH)2D3-to-25(OH)D3 both presupplementation and postsupplementation (P ≤ 0.005). Intergroup differences in 1α,25(OH)2D3-inducible gene expression signatures were modest and variable if statistically significant.Conclusions: Attenuation of the 25(OH)D response to vitamin D supplementation in asthma and COPD associated with reduced molar ratios of 25(OH)D3-to-vitamin D3 and increased molar ratios of 1α,25(OH)2D3-to-25(OH)D3 in serum, suggesting that vitamin D metabolism is dysregulated in these conditions.

Dysregulation of vitamin D synthesis pathway genes in colorectal cancer: A case-control study.

BACKGROUND: The cytochromes P450 are a superfamily of enzymes that control the synthesis of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3. These enzymes contribute to the formation of 1,25-dihydroxyvitamin D3, which starts with a 25-hydroxylation by CYP2R1 and CYP27A1 and a subsequent 1α-hydroxylation via CYP27B1. METHODS: By using quantitative real-time polymerase chain reaction (qRT-PCR), we analyzed the expression ratio of CYP2R1, CYP27A1 and CYP27B1 genes within the vitamin D metabolic pathway in a total of 75 colorectal cancer (CRC) tissues compared to the adjacent tissues. Furthermore, we evaluated the association of CYP27B1 rs4646536 and CYP2R1 rs12794714 and rs10766196 polymorphisms with CRC risk in a total of 490 subjects, including 245 CRC patients and 245 non-cancer controls. The genotyping was performed using tetra-primer amplification refractory mutation system polymerase chain reaction (TP-ARMS-PCR) method. RESULTS: The results indicated 2.3 and 2.7 upregulation of CYP2R1 and CYP27B1 genes in colorectal cancer tissues compared to the adjacent tissues, respectively. Rs12794714 AG genotype increased the risk of CRC (P = .03). Furthermore, a significant association was observed under the dominant inheritance model (P = .039). CONCLUSION: CYP2R1 and CYP27B1 genes were over-expressed in CRC samples compared to the adjacent control tissues. Furthermore, CYP2R1 rs12794714 variant was associated with the risk of CRC in the studied samples. CYP2R1 rs10766196 and CYP27B1 rs4646536 are not responsible for CYP2R1 and CYP27B1 genes expression alteration, respectively, but CYP2R1 rs12794714 polymorphism may be the reason of CYP2R1 upregulation and increased the risk of CRC.

Chlorpyrifos alters expression of enzymes involved in vitamin D3 synthesis in skin cells.

Skin acts as a mechanical barrier between human body and environment. Epidermal cells are regularly exposed to many physiological and environmental stressors, such as pesticides, like chlorpyrifos (CPS). It is recognised that CPS may affect metabolism of other exo- and endogenous substances by affecting enzyme activity and expression. This study aims to investigate the effect of CPS on expression of CYP27A1, CYP27B1 and CYP24A1, the enzymes involved in synthesis and metabolism of vitamin D3, in human keratinocytes HaCaT and human fibroblasts BJ. Synthesis of vitamin D3 in cells was initiated by irradiating with UVB. Expression of CYP27A1, CYP27B1 and CYP24A1 was evaluated by RT-qPCR and Western blot. Our experiments revealed that expression of all tested cytochrome P450 isoforms in cells exposed to CPS changed significantly. Exposure of HaCaT keratinocytes to CPS decreased CYP27A1 mRNA levels, but increased CYP27B1 and CYP24A1 mRNA levels. This was confirmed at the protein level, except for the CYP27A1 expression. Outcome for the BJ cells was however less conclusive. Though exposure to CPS decreased CYP27A1 and CYP27B1 mRNA levels, at protein level increasing concentration of CPS and UVB intensity induced expression of CYP27A1 and CYP24A1. The expression of CYP27B1 isoform decreased in line with mRNA level. Nevertheless, it can be concluded that CPS may therefore interrupt vitamin D3 metabolism in skin cells, but further studies are required to better understand such mechanisms.

Local expression profiles of vitamin D-related genes in airways of COPD patients.

Treatment of Chronic Obstructive Pulmonary Disease (COPD) is based on bronchodilation, with inhaled corticosteroids or azithromycin associated when frequent exacerbations occur. Despite the proven benefits of current treatment regimens, the need for new interventions in delineated subgroups remains. There is convincing evidence for oral vitamin D supplementation in reducing exacerbations in COPD patients severely deficient for circulating vitamin D. However, little is known about local vitamin D metabolism in the airways and studies examining expression of the vitamin D receptor (VDR), the activating enzyme (CYP27B1) and inactivating enzyme (CYP24A1) of vitamin D in lung tissue of COPD patients are lacking. Therefore, the expression and localization of key enzymes and the receptor of the vitamin D pathway were examined in tissue of 10 unused donor lungs and 10 COPD explant lungs. No differences in the expression of CYP27B1 and CYP24A1 were found. Although protein expression of VDR was significantly lower in COPD explant tissue, there was no difference in downstream expression of the antimicrobial peptide cathelicidin. Whereas CYP27B1 and CYP24A1 were present in all layers of the bronchial epithelium, VDR was only expressed at the apical layer of a fully differentiated bronchial epithelium with no expression in vascular endothelial cells. By contrast, CYP24A1 expression was highly present in lung endothelial cells suggesting that systemic vitamin D can be inactivated before reaching the epithelial compartment and the tissue immune cells. These data support the idea of exploring the role of vitamin D inhalation in patients with COPD.

Association study between vitiligo and autoimmune-related genes CYP27B1, REL, TNFAIP3, IL2 and IL21.

The aetiology of vitiligo has not been fully elucidated, and several hypotheses have been investigated; among them, the most explored assumes an autoimmune basis for the disease. Supporting this hypothesis is the frequent co-occurrence of autoimmune diseases with vitiligo. In addition, various genetic loci associated with vitiligo harbour key immune response genes. Our general hypothesis is that autoimmunity-associated genes participate in the control of vitiligo susceptibility. To investigate this hypothesis, we tested for association between vitiligo and genes CYP27B1, REL, TNFAIP3 and IL2/IL21, all previously related to autoimmune diseases associated with vitiligo. The study was performed using two independent population samples: a family-based discovery set (211 trios) and a replication set (131 cases/119 controls). Statistically significant association with vitiligo was detected between markers of the REL and IL2 gene in the family-based sample. Both association signals were concentrated among patients displaying autoimmune comorbidity and non-segmental vitiligo. Evidence for validation was detected for IL2 marker. Our findings suggest REL and IL2 as new vitiligo susceptibility genes and reinforce the hypothesis of a shared genetic mechanism controlling vitiligo and other autoimmune diseases.

Oral 25-Hydroxycholecalciferol Acts as an Agonist in the Duodenum of Mice and as Modeled in Cultured Human HT-29 and Caco2 Cells.

BACKGROUND: 25-Hydroxycholecalciferol [25(OH)D] is the predominant circulating metabolite of vitamin D and serves as the precursor for 1α,25-dihydroxycholecalciferol [1,25(OH)2D], the hormonally active form. The presence of 1α-hydroxylase (1α-OHase) in the intestine suggests that 1,25(OH)2D can be produced from 25(OH)D, but the effects of oral 25(OH)D on the intestine have not been determined. OBJECTIVES: We investigated the acute intestinal response to orally consumed 25(OH)D in mice by assessing mRNA induction of cytochrome p450 family 24 subfamily A member 1 (Cyp24), a vitamin D-dependent gene. The mechanism of action then was determined through in vitro analyses with Caco2 and HT-29 cells. METHODS: Adult male C57BL6 mice were given a single oral dose of 40, 80, 200, or 400 ng 25(OH)D (n = 4 per dose) or vehicle (n = 3), and then killed 4 h later to evaluate the duodenal expression of Cyp24 mRNA by qPCR and RNA in situ hybridization. The 25(OH)D-mediated response was also evaluated with Caco2 and HT-29 cells by inhibition assay and dose-response analysis. A cytochrome p450 family 27 subfamily B member 1 (CYP27B1) knockdown of HT-29 was created to compare the dose-response parameters with wild-type HT-29 cells. RESULTS: Oral 25(OH)D induced expression of Cyp24 mRNA in the duodenum of mice with 80 ng 25(OH)D by 3.3 ± 0.8 ΔΔCt compared with controls (P < 0.05). In vitro, both Caco2 and HT-29 cells responded to 25(OH)D treatment with 200-fold and 175-fold greater effective concentration at 50% maximal response than 1,25(OH)2D, yet inhibition of 1α-OHase and knockdown of CYP27B1 had no effect on the responses. CONCLUSIONS: In mice, orally consumed 25(OH)D elicits a vitamin D-mediated response in the duodenum. In vitro assessments suggest that the response from 25(OH)D does not require activation by 1α-OHase and that 25(OH)D within the intestinal lumen acts as a vitamin D receptor agonist.

Vitamin D in Type 2 Diabetes: Genetic Susceptibility and the Response to Supplementation.

Variants of vitamin D metabolism-genes may predispose to type 2 diabetes (T2D). This study investigated the impact of these variants on disease susceptibility, Vitamin D, parathyroid hormone, C-peptide and HbA1c levels before and after cholecalciferol supplementation in patients with T2D.Twelve polymorphisms within CYP2R1, CYP27B1, DBP, VDR and CYP24A1 were genotyped in 553 T2D patients and 916 controls. In addition 65 patients receiving either cholecalciferol or placebo were analyzed during 6 months intervention and 6 months follow-up.T2D risk alleles are VDR rs7975232 "G" (pc=0.031), rs1544410 "G" (pc=0.027) and CYP2R1 rs10741657 "A" (pc=0.016). Patients with genotypes CYP27B1 rs10877012 "CC" (pc=4x10-5), DBP rs7041 "GG" (pc=0.003), rs4588 "CC" (pc = 3x10-4), CYP24A1 rs2585426 "CG" (pc=0.006) and rs2248137 "CG" (pc=0.001) showed lower 25(OH)D3 and DBP rs4588 "CC" lower 1,25(OH)2D3 levels (pc=0.005). Whereas DBP rs4588 "CC" (pc=0.009), CYP27B1 rs10877012 "AC" (pc=0.059), VDR rs7975323 "AG" (pc=0.033) and rs1544410 "GG" (pc=0.013) are associated with higher 25(OH)D3 levels at 6 months' follow-up. Significant PTH suppression was detected for CYP2R1 "AG" (pc=0.002), DBP rs4588 "CC" (pc<0.001), VDR rs110735810 "CT" (pc<0.001) and CYP24A1 rs2248137 "GG" (pc=0.021).Genetic variants of the vitamin D system predispose to type 2 diabetes and regulate - partially - vitamin D metabolism, concentrations and the vitamin D status. Vitamin D insufficiency is a T2D risk factor. The response to cholecalciferol supplementation can be measured as 25(OH)D3 increment and PTH suppression. This process is regulated by genes of the vitamin D system conferring modest T2D risk.

Association of genetic variations in the vitamin D pathway with susceptibility to tuberculosis in Kazakhstan.

Tuberculosis (TB) poses an important health challenge and a significant economic burden for Kazakhstan and in Central Asia. Recent findings show a number of immunological related processes and host Mycobacterium tuberculosis defense are impacted by a variety of genes of the human host including those that play a part in the vitamin D metabolism. We investigated the genetic variation of genes in the vitamin D metabolic pathway of a cohort 50 TB cases in Kazakhstan and compared them to 34 controls living in the same household with someone infected with TB. We specifically analyzed 11 SNPs belonging to the following genes: DHCR7, CYP2R1, GC-1, CYP24A1, CYP27A1, CYP27B1, VDR and TNFα. These genes play a number of different roles including synthesis, activation, delivery and binding of the activated vitamin D. Our preliminary results indicate significant association of VDR (vitamin D receptor) SNPs (rs1544410, BsmI, with OR = 0.425, CI 0.221-0.816, p = 0.009 and rs731236, TaqI with OR = 0.443, CI 0.228-0.859, p = 0.015) and CYP24A1 (rs6013897 with OR = 0.436, CI 0.191-0.996, p = 0.045) with TB. Interaction of genetic variation of VDR and CYP24A1 may impact susceptibility to TB. The findings provided initial clues to understand individual genetic differences in relation to susceptibility and protection to TB.

Association of Vitamin D Metabolism Gene Polymorphisms with Autoimmunity: Evidence in Population Genetic Studies.

A high prevalence of vitamin D (calcidiol) serum deficiency has been described in several autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (AR), and systemic lupus erythematosus (SLE). Vitamin D is a potent immunonutrient that through its main metabolite calcitriol, regulates the immunomodulation of macrophages, dendritic cells, T and B lymphocytes, which express the vitamin D receptor (VDR), and they produce and respond to calcitriol. Genetic association studies have shown that up to 65% of vitamin D serum variance may be explained due to genetic background. The 90% of genetic variability takes place in the form of single nucleotide polymorphisms (SNPs), and SNPs in genes related to vitamin D metabolism have been linked to influence the calcidiol serum levels, such as in the vitamin D binding protein (VDBP; rs2282679 GC), 25-hydroxylase (rs10751657 CYP2R1), 1α-hydroxylase (rs10877012, CYP27B1) and the vitamin D receptor (FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236) VDR). Therefore, the aim of this comprehensive literature review was to discuss the current findings of functional SNPs in GC, CYP2R1, CYP27B1, and VDR associated to genetic risk, and the most common clinical features of MS, RA, and SLE.

Tuning Adipogenic Differentiation in ADSCs by Metformin and Vitamin D: Involvement of miRNAs.

Fat tissue represents an important source of adipose-derived stem cells (ADSCs), which can differentiate towards several phenotypes under certain stimuli. Definite molecules as vitamin D are able to influence stem cell fate, acting on the expression of specific genes. In addition, miRNAs are important modulating factors in obesity and numerous diseases. We previously identified specific conditioned media able to commit stem cells towards defined cellular phenotypes. In the present paper, we aimed at evaluating the role of metformin on ADSCs differentiation. In particular, ADSCs were cultured in a specific adipogenic conditioned medium (MD), in the presence of metformin, alone or in combination with vitamin D. Our results showed that the combination of the two compounds is able to counteract the appearance of an adipogenic phenotype, indicating a feedforward regulation on vitamin D metabolism by metformin, acting on CYP27B1 and CYP3A4. We then evaluated the role of specific epigenetic modulating genes and miRNAs in controlling stem cell adipogenesis. The combination of the two molecules was able to influence stem cell fate, by modulating the adipogenic phenotype, suggesting their possible application in clinical practice in counteracting uncontrolled lipogenesis and obesity-related diseases.

Vitamin D Status Modulates Inflammatory Response in HIV+ Subjects: Evidence for Involvement of Autophagy and TG2 Expression in PBMC.

Conflicting results on the involvement of vitamin D deficiency in inflammatory and immune response in HIV+ subjects are reported. We aimed to characterize the possible influence of vitamin D status on changes in expression of tissue transglutaminase gene (TGM2) and other genes involved in inflammatory response and autophagy in peripheral blood mononuclear cells (PBMC) from HIV+ subjects. HIV+ subjects (n = 57) under antiretroviral therapy (ART) and healthy controls (n = 40) were enrolled. mRNA levels of 1-alpha-hydroxylase (CYP27B1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), TGM2, microtubule-associated protein 1A/1B-light chain 3 (LC3), autophagy-related 5 homolog (ATG5), and Beclin 1 (BECN1) were quantified by real-time PCR. In HIV+ subjects, 25(OH)D3 plasma levels were negatively correlated with time since HIV diagnosis. In PBMC from HIV+ subjects, increases in gene expression of TNF-α and IFN-γ in comparison to controls were observed. The highest increase in TNF-α transcripts was observed in HIV+ subjects with deficient 25(OH)D3 levels. Autophagy-related genes LC3, ATG5, and BECN1 were down-regulated in HIV+ subjects. Moreover, TGM2 transcripts were up-regulated in PBMC from HIV+ subjects with 25(OH)D3 deficiency. Changes observed in PBMC from HIV+ subjects appeared to be dependent on vitamin D status. The present results suggest that vitamin D deficiency is associated with changes in the expression of markers of inflammation and autophagy, resulting in immune cell dysfunction.