Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity,Vitamin D and FokI

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D₃ status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D₃ levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)₂D₃. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D₃ status was normal and not significantly different between ethnicities. Neither 25(OH)D₃ status nor 1,25(OH)₂D₃ supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)₂D₃-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D₃ status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)₂D₃-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.     

Effects of Vitamin D on Airway Epithelial Cell Morphology and Rhinovirus Replication

Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)₂D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0–69.0] vs. 30 µm [24.5–34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1β) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells.     

Vitamin D Receptor Gene Polymorphisms Are Associated with Obesity and Inflammosome Activity

To explore the mechanisms underlying the suggested role of the vitamin D/vitamin D receptor (VDR) complex in the pathogenesis of obesity we performed genetic and immunologic analyses in obese and non-obese Saudi individuals without other concomitant chronic diseases. Genomic DNA was genotyped for gene single nucleotide polymorphisms (SNPs) of VDR by allelic discrimination in 402 obese (body mass index –BMI≥30 kg/m2) and 489 non-obese (BMI<30 kg/m2) Saudis. Q-PCR analyses were performed using an ABI Prism 7000 Sequence Detection System. The inflammosome pathway was analysed by PCR, cytokines and plasma lipopolysaccaride (LPS) concentrations with ELISA assays. Results showed that the VDR SNPs rs731236 (G) (TaqI) and rs1544410 (T) (Bsm-I) minor allele polymorphisms are significantly more frequent in obese individuals (p = 0.009, β = 0.086 and p = 0.028, β = 0.072, respectively). VDR haplotypes identified are positively (GTA) (p = 0.008, β = 1.560); or negatively (ACC) (p = 0.044, β = 0.766) associated with obesity and higher BMI scores. The GTA "risk" haplotype was characterized by an up-regulation of inflammosome components, a higher production of proinflammatory cytokines (p<0.05) and a lower VDR expression. Plasma LPS concentration was also increased in GTA obese individuals (p<0.05), suggesting an alteration of gut permeability leading to microbial translocation. Data herein indicate that polymorphisms affecting the vitamin D/VDR axis play a role in obesity that is associated with an ongoing degree of inflammation, possibly resulting from alterations of gut permeability and microbial translocation. These results could help the definition of VDR fingerprints that predict an increased risk of developing obesity and might contribute to the identification of novel therapeutic strategies for this metabolic condition.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

1,25-Dihydroxyvitamin D and the Vitamin D Receptor Gene Polymorphism Apa1 Influence Bone Mineral Density in Primary Hyperparathyroidism

Objective

Parathyroid hormone (PTH) and vitamin D are the most important hormones regulating calcium metabolism. In primary hyperparathyroidism (PHPT) excessive amounts of PTH are produced. Bone turnover is enhanced, leading to reduced bone mineral density and elevated levels of serum calcium. The aim of this study was to investigate relations between serum levels of 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)₂D) and bone mineral density, as well as known genetic polymorphisms in the vitamin D receptor and enzymes metabolising vitamin D in patients with PHPT.

Design/Subjects

We conducted a cross-sectional study of 52 patients with PHPT.

Results

Mean level of 25(OH)D was 58.2 nmol/L and median 1,25(OH)₂D level was 157 pmol/L. Among our patients with PHPT 36.5% had 25(OH)D levels below 50 nmol/L. Serum 1,25(OH)₂D was inversely correlated to bone mineral density in distal radius (p = 0.002), but not to bone mineral density at lumbar spine or femoral neck. The vitamin D receptor polymorphism Apa1 (rs7975232) was associated with bone mineral density in the lumbar spine.

Conclusions

The results suggest that PHPT patients with high blood concentrations of 1,25(OH)₂D may have the most deleterious skeletal effects. Randomized, prospective studies are necessary to elucidate whether vitamin D supplementation additionally increases serum 1,25(OH)₂D and possibly enhances the adverse effects on the skeleton in patients with PHPT.     

Recruitment of cAMP-response Element-binding Protein and Histone Deacetylase Has Opposite Effects on Glucocorticoid Receptor Gene Transcription

Glucocorticoids control the synthesis of the glucocorticoid receptor (GR) in various tissues through a negative feedback regulation of the mRNA. In this study, we have identified feedback regulatory domains in the human GR gene promoter and examined the roles of GR, the cAMP-response element-binding protein (CREB), and HDAC-6 in association with promoter elements of the human GR gene. Using breast cancer T47D and HeLa-GR cells, we identify specific negative glucocorticoid-response elements in the GR gene. The feedback regulatory domains were also involved in interactions with CREB. GR-bound negative glucocorticoid-response elements recruited HDAC-6, and this was dependent on treatment with dexamethasone. Both CREB and HDAC-6 formed complexes with GR-dexamethasone. The HDAC-6 LXXLL motif between amino acids 313 and 418 made direct contact with the GR AF-1 domain. Interestingly enough, although the level of GR decreased in CREB knockdown cells, it was elevated in HDAC-6 knockdown cells. Our results suggest that CREB-P is dephosphorylated and that HDAC-6 is recruited by the GR, and they play opposite roles in the negative feedback regulation of the GR gene.     

The Role of the rs1544410 Polymorphism of Vitamin D Receptor Gene in Breast Cancer Susceptibility

This study was devised to investigate the genetic effect modification of the BsmI polymorphism associated with the susceptibility to breast cancer. Case–control studies of the BsmI polymorphism and breast cancer were searched. A total of 17 eligible publications were included in our final analysis. Pooled ORs and 95 % CIs were obtained by means of fixed effects model. The general and stratified analyses according to ethnicity showed that the association between the BsmI polymorphism and the risk of breast cancer was not statistically significant. However, the subgroup of the hospital-based studies was found to confer protection against the disease (OR_BBvs.bb = 0.83, 95 % CI = 0.71–0.97, P _h = 0.571; OR_BBvs.Bb+bb = 0.86, 95 % CI = 0.74–1.00, P _h = 0.903; OR_{allele B vs. allele b} = 0.92, 95 % CI = 0.86–0.99, P _h = 0.337). Our results suggested that the presence of the BsmI polymorphism may contribute to the susceptibility of breast cancer. It is necessary that future large-scale studies should be conducted to further confirm the association between the BsmI polymorphism and breast cancer risk.     

Vitamin D Receptor Gene Polymorphisms and Prognosis of Breast Cancer among African-American and Hispanic Women

Background

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts.

Methods

Blood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method.

Results

An increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome.

Conclusions

VDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer.     

维生素D受体最新研究进展

维生素D受体(vitamin D receptor,VDR)是一种核转录因子,通过与配体特异结合,调控多种基因的表达,从而调节多种生命活动的进行.本文总结了近年来VDR的研究进展,主要包括VDR的作用机制、VDR行使功能所需共激活子及共抑制子、VDR在生长分化、免疫调节和抑制肿瘤等方面的作用及VDR配体类似物在药物开发研究领域的最新进展.     

Investigation on the Potential Role of the AH Receptor Nuclear Translocator Protein in Vitamin D Receptor Action

Abstract

The Ah receptor nuclear translocator protein (ARNT) is required for binding of the Ah (dioxin) receptor to the xenobiotic responsive element (XRE), and is a structural component of the XRE-binding form of the Ah receptor. The vitamin D receptor requires an accessory protein for binding to the vitamin D responsive element (VDRE) in the osteocalcin gene. Since the vitamin D receptor has similarities to the Ah receptor, we investigated whether ARNT is also required for vitamin D receptor activity. Two lines of evidence demonstrate that ARNT is not required for vitamin D receptor activity, and therefore does not correspond to the vitamin D receptor accessory protein: i) Antibodies to ARNT have no effect on binding of the vitamin D receptor to the VDRE. ii) c4, a mutant of Hepa-1 cells that is defective in ARNT activity, and in which binding of the Ah receptor to the XRE does not occur, possesses a vitamin D receptor with full activity for binding the VDRE.     

Significance of Vitamin D Receptor Gene Polymorphisms for Risk of Hepatocellular Carcinoma in Chronic Hepatitis C

BACKGROUND/AIMSBiological and epidemiological data suggest that vitamin D levels may influence cancer development. Several single nucleotide polymorphisms have been described in the vitamin D receptor (VDR) gene in association with cancer risk. We aimed to investigate the association of VDR gene polymorphisms with hepatocellular carcinoma (HCC) development in chronic hepatitis C patients.METHODSIn a cross-sectional, hospital-based setting, 340 patients (201 chronic hepatitis, 47 cirrhosis and 92 HCC) and 100 healthy controls receiving VDR genotyping (bat-haplotype: BsmI rs1544410 C, ApaI rs7975232 C and TaqI rs731236 A) were enrolled.RESULTSPatients with HCC had a higher frequency of ApaI CC genotype (P = 0.027) and bAt[CCA]-haplotype (P = 0.037) as compared to control subjects. There were no differences in BsmI and TaqI polymorphisms between two groups. In patients with chronic hepatitis C, HCC subjects had a higher frequency of ApaI CC genotype and bAt[CCA]-haplotype than those with chronic hepatitis (P = 0.001 and 0.002, respectively) and cirrhosis (P = 0.019 and 0.026, respectively). After adjusting age and sex, logistic regression analysis showed that ApaI CC genotype (odds ratio: 3.02, 95% confident interval: 1.65-5.51) was independently associated with HCC development.CONCLUSIONVDR ApaI polymorphism plays a role in the development of HCC among chronic hepatitis C patients. Further explorations of this finding and its implications are required.     

Association between Vitamin D Receptor Gene Polymorphisms and Breast Cancer Risk: A Meta-Analysis of 39 Studies

Background

The associations between vitamin D receptor (VDR) gene polymorphisms and breast cancer risk were comprehensively investigated to clarify issues that remain controversial.

Methodology/Principal Findings

An electronic search was conducted of several databases, including PubMed, the Cochrane library, Web of Science, EMBASE, CBM and CNKI, for papers that describe the association between Fok1, poly-A repeat, Bsm1, Taq1 or Apa1 polymorphisms of the VDR gene and breast cancer risk. Summary odds ratios and 95% confidence intervals (CI) were estimated based on a fixed-effect model (FEM) or random-effect model (REM), depending on the absence or presence of significant heterogeneity. A total of 39 studies met the inclusion criteria. A meta-analysis of high-quality studies showed that the Fok1 polymorphism of the VDR gene was associated with an increased risk of breast cancer (ff vs. Ff+FF, OR: 1.09, 95%CI: 1.02 to 1.16, p = 0.007). No significant associations were observed between the other polymorphisms and breast cancer risk. No positive results were detected by pooling the results of all relevant studies.

Conclusion

A meta-analysis of high-quality studies demonstrated that the Fok1 polymorphism of the VDR gene was closely associated with breast cancer risk.     

Vitamin D,Vitamin D Binding Protein,and Longitudinal Outcomes in COPD

Background

Associations between Vitamin D₃ [25(OH)D], vitamin D binding protein (VDBP) and chronic obstructive pulmonary disease (COPD) are previously reported. We aimed to further investigate these associations on longitudinal outcomes.

Methods

426 COPD patients from western Norway, GOLD stage II-IV, aged 40–76, were followed every six-month from 2006 through 2009 with spirometry, bioelectrical impedance measurements and registration of exacerbation frequency. Serum 25(OH)D and VDBP levels were determined at study-entry by high-performance liquid chromatography coupled with mass spectrometry and enzyme immunoassays respectively. Yearly change in lung function and body composition was assessed by generalized estimating equations (GEE), yearly exacerbation rate by negative binomial regression models, and 5 years all-cause mortality by Cox proportional-hazard regression.

Results

1/3 of the patients had vitamin D deficiency (<20ng/mL) and a greater decline in both FEV1 and FVC, compared to patients with normal levels; for FEV1 this difference only reached statistical significance in the 28 patients with the lowest levels (<10ng/mL, p = 0.01). Neither 25(OH)D nor VDBP levels predicted exacerbation rate, change in fat free mass index or risk of death.

Conclusion

Severe vitamin D deficiency may affect decline in lung function parameters in COPD. Neither 25(OH)D nor VDBP levels did otherwise predict markers of disease progression.     

Epicardial Ablation on the Beating Heart: Limited Efficacy of a Novel, Cooled Radiofrequency Ablation Device

OBJECTIVE: To perform a minimally invasive procedure for atrial fibrillation without cardiopulmonary bypass, it is necessary to create transmural lesions on the beating heart. Although bipolar radiofrequency clamps can isolate the pulmonary veins, they have difficulty in performing any other left atrial lesions, particularly those of the traditional Cox-Maze procedure. This study examined the performance of an internally cooled, bipolar radiofrequency device designed for such an application. METHODS: Eleven domestic pigs underwent median sternotomy. Five animals had eight atrial lesions created with the radiofrequency device at times of 20, 30, 40, and 50 seconds. In six other pigs, the device was compared with another technology that has been used clinically for epicardial, beating heart ablation, the Guidant Flex 4 microwave device. The tissue was stained with 2,3,5-triphenyl-tetrazoluim chloride, and the lesions were sectioned at 5-mm intervals. Lesion width, depth, and transmurality were evaluated. RESULTS: The radiofrequency device had a linear dose-response relationship. Lesions were wider and deeper with increasing ablation times. A total of 40%, 45%, 60%, and 67% of lesions were transmural at times of 20, 30, 40, and 50 seconds, respectively. Ninety-one percent of lesions in tissue up to 4-mm thick were transmural after 50 seconds. However, performance in thicker tissue was poor. Lesions created by the device were deeper and more likely to be transmural than the Flex 4. CONCLUSIONS: This internally cooled, bipolar radiofrequency device can reliably create transmural lesions on tissue up to 4-mm thick and performs better than a microwave device.     

Action of Vitamin D and the Receptor,VDRa, in Calcium Handling in Zebrafish (Danio rerio)

The purpose of the present study was to use zebrafish as a model to investigate how vitamin D and its receptors interact to control Ca²⁺ uptake function. Low-Ca²⁺ fresh water stimulated Ca²⁺ influx and expressions of epithelial calcium channel (ecac), vitamin D-25-hydroxylase (cyp2r1), vitamin D receptor a (vdra), and vdrb in zebrafish. Exogenous vitamin D increased Ca²⁺ influx and expressions of ecac and 25-hydroxyvitamin D₃-24-hydroxylase (cyp24a1), but downregulated 1α-OHase (cyp27b1) with no effects on other Ca²⁺ transporters. Morpholino oligonucleotide knockdown of VDRa, but not VDRb, was found as a consequence of calcium uptake inhibition by knockdown of ecac, and ossification of vertebrae is impaired. Taken together, vitamin D-VDRa signaling may stimulate Ca²⁺ uptake by upregulating ECaC in zebrafish, thereby clarifying the Ca²⁺-handling function of only a VDR in teleosts. Zebrafish may be useful as a model to explore the function of vitamin D-VDR signaling in Ca²⁺ homeostasis and the related physiological processes in vertebrates.     

Polymorphisms in the Calcium-Sensing Receptor Gene Are Associated with Clinical Outcome of Neuroblastoma

Background

Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) to assess if genetic variants producing a less active receptor are associated with more aggressive disease course.

Methods

Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays.

Results

Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002) and the histological subgroup of undifferentiated neuroblastomas (P = 0.046). Patients harboring this polymorphism had significantly lower overall (P = 0.022) and event-free survival (P = 0.01) rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14–5.29]; P = 0.022) and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20–6.25]; P = 0.016).

Conclusions

The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636 polymorphism. Thus, its association with metastatic disease and poor outcome would add to our previous data and further support that inactivation of the CaSR gene is a mechanism associated with neuroblastoma malignant behavior.     

Placental and Embryonic Growth Restriction in Mice With Reduced Function Epidermal Growth Factor Receptor Alleles

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr^wa2 allele or heterozygous for the antimorphic Egfr^Wa5 allele. Egfr^wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr^Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr^wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr^Wa5 heterozygotes than in Egfr^wa2 homozygotes and suggest that Egfr^wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to strain-dependent differences in intrauterine growth restriction caused by reduced EGFR activity.EPIDERMAL growth factor receptor (EGFR) is the prototypical member of the ERBB family of receptor tyrosine kinases and is known to regulate many aspects of cellular biology including cell proliferation, survival, differentiation, and migration (reviewed in Yarden and Sliwkowski 2001). Eleven known ligands bind the extracellular region of ERBB-family receptors, and activation of the tyrosine kinase domain occurs following receptor homo- or heterodimerization. The resulting biological responses are dependent upon specific signaling cascades initiated by ERBBs and can be influenced by the particular ligand–ERBB combination (Yarden and Sliwkowski 2001). Studies using cultured cells have underscored the importance of EGFR in modulating various cellular processes, while animal models have been able to demonstrate that EGFR is required for numerous developmental and physiological processes (Casalini et al. 2004). In vivo studies have shown that EGFR is particularly important for normal placental development in mice; placentas from Egfr nullizygous (Egfr^{tm1Mag/tm1Mag}) embryos exhibit strain-specific defects that result in differential embryonic lethality (Sibilia and Wagner 1995; Threadgill et al. 1995). Two additional Egfr alleles result in reduced EGFR signaling in mice: the recessive hypomorphic Egfr^wa2 and dominant antimorphic Egfr^Wa5 alleles (Luetteke et al. 1994; Fowler et al. 1995; Du et al. 2004; Lee et al. 2004). These alleles can provide insight into the level of EGFR signaling required for normal placental development.Egfr^wa2 is a classical spontaneous mutation that arose in 1935 that causes a distinct wavy coat phenotype in the homozygote (Figure 1; Keeler 1935). This recessive mutation was subsequently found to be a single nucleotide transversion resulting in a valine → glycine substitution in the highly conserved kinase domain of EGFR (Luetteke et al. 1994; Fowler et al. 1995). Since mice homozygous for the Egfr^tm1Mag null allele die before or shortly after birth depending on genetic background, the hypomorphic Egfr^wa2 allele has been the primary model used to study the effect of attenuated EGFR signaling in a variety of adult physiological and disease states. In addition to eye and hair phenotypes, the adult Egfr^wa2 homozygous mouse exhibits delayed onset of puberty, abnormal ovulation, enlarged aortic valves and cardiac hypertrophy, decreased body size, defects in mammary gland development and lactation, increased susceptibility to colitis, and impaired intestinal adaptation following small bowel resection (Fowler et al. 1995; Helmrath et al. 1997; Chen et al. 2000; Egger et al. 2000; O''Brien et al. 2002; Prevot et al. 2005; Hsieh et al. 2007). Despite the widespread use of the Egfr^wa2 allele, there are limitations in using Egfr^wa2 homozygous mice to clearly define the physiological roles of EGFR. Egfr^wa2 has traditionally been maintained in cis, tightly linked with a hypomorphic Wnt3a allele, Wnt3a^vt (vestigal tail), making phenotypic analysis of reduced EGFR signaling by itself difficult. Furthermore, Egfr^wa2 has also typically been maintained on a mixed genetic background and since the Egfr nullizygous phenotype is similarly influenced by genetic modifiers, a mixed background could mask phenotypes that become evident when Egfr^wa2 mice are inbred.Open in a separate windowFigure 1.—Congenic 129 Egfr allelic series. Wild-type (left), Egfr^wa2 homozygote (middle), and Egfr^wa5 heterozygote (right) mice. As weanlings and adults, the Egfr^wa2 homozygotes and Egfr^wa5 heterozygotes are grossly indistinguishable.The Egfr^Wa5 allele arose in a large, genomewide N-ethyl-N-nitrosourea mutagenesis screen for dominant visible mutations in the mouse. Egfr^Wa5 heterozygous mice were first identified by their open eyelids at birth and by development of a wavy coat, similar to the phenotype of Egfr^wa2 homozygous mice (Figure 1). Egfr^Wa5 failed to complement the Egfr^tm1Mag null allele and was shown to function as an antimorph since Egfr^Wa5, but not Egfr^tm1Mag, heterozygotes exhibit eyelid and coat phenotypes (Lee et al. 2004). A single nucleotide missense mutation was found in the Egfr^Wa5 allele that results in an Asp → Gly substitution in the highly conserved DFG domain of the EGFR kinase catalytic loop (Du et al. 2004; Lee et al. 2004). Although Egfr^Wa5 heterozygotes are viable, Egfr^Wa5 homozygotes die prenatally and exhibit placental defects identical to those from Egfr^tm1Mag homozygous null embryos. Placentas from Egfr^Wa5 heterozygotes on a mixed background show variable reduction in the spongiotrophoblast layer and minor abnormalities in the labyrinth region, but no effects on embryo survival have been reported.In vitro studies with Egfr^Wa5 suggest that it encodes a kinase-dead EGFR since no phosphorylation of EGFR^Wa5 is detected following stimulation with ligands. In agreement with the genetic data showing that Egfr^Wa5 is an antimorph, in vitro studies have demonstrated that the EGFR^Wa5 receptor can inhibit phosphorylation of EGFR and MAPK in a dose-dependent manner (Lee et al. 2004). In Chinese hamster ovary cells expressing an equimolar ratio of EGFR and EGFR^Wa5 receptors, <10% of wild-type phosphorylation levels were observed by Western blot analysis.The Egfr allelic series available in the mouse has high utility for studying gene function since EGFR is involved in a multitude of developmental processes and human diseases. Although both Egfr^wa2 and Egfr^Wa5 alleles result in reduced EGFR signaling, the activity and phenotypic consequences of Egfr^wa2 homozygosity has not been compared to that of Egfr^Wa5 heterozygosity when both are on the same genetic backgrounds. Adult Egfr^Wa5 heterozygous mice appear highly similar to Egfr^wa2 homozygotes, but crosses with the Apc^Min intestinal tumor model have shown that a more substantial reduction in tumor number occurs when the Apc^Min mutation is bred onto the Egfr^wa2 homozygous vs. Egfr^Wa5 heterozygous background (Roberts et al. 2002; Lee et al. 2004). These results suggest that Egfr^Wa5 heterozygous mice retain higher levels of EGFR activity than Egfr^wa2 homozygous mice; however, the data are confounded by the fact that the crosses were performed using different mixed genetic backgrounds.This study reports a comprehensive genetic analysis of reduced EGFR signaling in Egfr^wa2 homozygotes and Egfr^Wa5 heterozygotes in placental development and embryonic growth for three congenic backgrounds, C57BL/6J (B6), 129S1/SvImJ (129), and BTBR/J-T+, tf/tf (BTBR). Wild-type placenta weight, embryo weight, and mRNA levels of genes selected for their trophoblast-specific expression were found to be highly strain dependent. Egfr^wa2 homozygous placentas are reduced in size in all three strains, and a proportion of 129-Egfr^wa2 homozygotes die before 15.5 days post-coitus (dpc). Egfr^wa2 homozygous embryos also display background-dependent intrauterine growth restriction (IUGR) in late gestation, which is most severe on 129 and BTBR backgrounds and models EGFR-associated IUGR in humans. Egfr^Wa5 heterozygous placentas exhibit a minor reduction in size on all three backgrounds with no impact on embryonic growth. These results suggest that reduced levels of EGFR signaling can interfere with normal placental development and that embryo development is affected only after placental size is sufficiently reduced. In addition, our data show that the level of EGFR signaling in Egfr^Wa5 heterozygous mice is higher than in Egfr^wa2 homozygotes and suggests that different Egfr allele combinations can be generated to “genetically titer” total EGFR activity in vivo.     

Vitamin D Receptor Gene FOKI Polymorphism Contributes to Increasing the Risk of HIV-Negative Tuberculosis: Evidence from a Meta-Analysis

BackgroundVitamin D receptor (VDR) gene FokI polymorphism have been studied in relation to tuberculosis (TB) in many populations and provided inconsistent results. In this study, we carried out a meta-analysis to derive a more reliable assessment on FokI polymorphism and the risk of HIV-negative TB.MethodsThe Embase, PubMed, and Cochrane Library databases were used to undertake a comprehensive systematic literature review of all current published VDR gene FOKI association studies aimed at the risk of TB up to June 30, 2015. Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were used to measure the strength of the models.ResultsA total of 14 studies (1,668 cases and 1,893 controls) were retrieved in the meta-analysis. The pooled OR was 1.60 (95% = 1.28–1.97, P<0.001; I ² = 29.5%, and P = 0.141 for heterogeneity) in the best genetic model (recessive model: ff vs. fF+FF). In the subgroup analysis by ethnicities, a significantly increased risk was found in the Asian group (OR = 1.82, 95% CI = 1.42–2.33, P<0.001; I ² = 31.0%, and P = 0.150 for heterogeneity) in the recessive model. Similarly, significant associations were also found in the polymerase chain reaction-restriction fragment length polymorphism group, high-quality studies, and the population based or hospital based groups. Moderate heterogeneity was found in this study.ConclusionOur results suggested that VDR FokI polymorphism contributes to increasing the risk of TB in HIV-negative individuals, especially in the Asian region. Further studies on this topic in other races are expected to be conducted in future.