FokI-RYdCas9 Mediates Nearly PAM-Less and High-Precise Gene Editing in Human Cells.

The demand for high-precision CRISPR/Cas9 systems in biomedicine is experiencing a notable upsurge. The editing system fdCas9 employs a dual-sgRNA strategy to enhance editing accuracy. However, the application of fdCas9 is constrained by the stringent requirement for two protospacer adjacent motifs (PAMs) of Cas9. Here, we devised an optimized editor, fRYdCas9, by merging FokI with the nearly PAM-less RYdCas9 variant, and two fRYdCas9 systems formed a dimer in a proper spacer length to accomplish DNA cleavage. In comparison to fdCas9, fRYdCas9 demonstrates a substantial increase in the number of editable genomic sites, approximately 330-fold, while maintaining a comparable level of editing efficiency. Through meticulous experimental validation, we determined that the optimal spacer length between two FokI guided by RYdCas9 is 16 base pairs. Moreover, fRYdCas9 exhibits a near PAM-less feature, along with no on-target motif preference via the library screening. Meanwhile, fRYdCas9 effectively addresses the potential risks of off-targets, as analyzed through whole genome sequencing (WGS). Mouse embryonic editing shows fRYdCas9 has robust editing capabilities. This study introduces a potentially beneficial alternative for accurate gene editing in therapeutic applications and fundamental research.

Molecular investigation of vitamin D receptor (VDR) genetic variants and their impact on VDR mRNA and serum vitamin D levels in allergic rhinitis in an Indian population: A case-control study.

Vitamin D deficiency is widespread and poses a significant health concern, as emerging research links it to allergic diseases owing to its immunomodulatory functions. The optimal functioning of vitamin D and its activation depend on its nuclear receptor, vitamin D receptor (VDR). Genetic variants of VDR have been explored as potential factors in autoimmune and allergic diseases, with limited studies on their association with allergic rhinitis (AR). The present investigation aims to analyse the role of three VDR genetic variants - TaqI, FokI and BsmI - in AR susceptibility and their impact on VDR mRNA and serum vitamin D levels. A total of 550 subjects, consisting of 250 AR cases and 300 age- and gender-matched controls, underwent genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). VDR mRNA and vitamin D levels were determined by quantitative real-time PCR and chemiluminescence, respectively. Although TaqI did not exhibit significant differences, FokI demonstrated a noteworthy association with AR, particularly with the CC genotype (odds ratio [OR]: 3.34; confidence interval [CI]: 1.79-6.23). Similarly, BsmI revealed an increased risk for AR, with the GA + AA genotypes showing a 2.2-fold elevated risk (OR: 2.20; CI: 1.53-3.16). VDR mRNA expression was threefold lower in AR patients (p < .0001), accompanied by reduced serum vitamin D levels (p < .0001). In addition, CC (p = .01) and AA (p = .02) genotypes of FokI and BsmI were associated with reduced VDR mRNA levels, whereas TaqI showed no such association. Similarly, heterozygous genotypes of TaqI and FokI, as well as homozygous AA of BsmI, correlated with lower serum vitamin D levels (p < .001). This study emphasizes the intricate relationship among VDR genetic variations, altered VDR activity, immune modulation and vitamin D metabolism in AR. Further research involving diverse populations is crucial for confirming and generalizing these findings, paving the way for personalized therapeutic interventions in vitamin D-related disorders.

Vitamin D receptor gene polymorphisms role in COVID-19 severity: Results of a Mexican patients' cohort.

Vitamin D status has been involved with coronavirus disease 19 (COVID-19) severity. This may be mediated by vitamin D metabolism regulatory genes. Of interest is the vitamin D receptor (VDR) gene, which has been previously associated with other inflammatory and respiratory diseases. In order to investigate the role of VDR gene polymorphisms in COVID-19 severity and outcome, a total of 292 COVID-19 patients were classified according to severity in moderate (n = 56), severe (n = 89) and critical (n = 147) and, according to outcome in survivor (n = 163) and deceased (n = 129), and analysed for FokI and TaqI VDR gene polymorphisms by polymerase chain reaction-based restriction enzyme digestion. The FokI and TaqI single nucleotide polymorphisms (SNPs) were not associated with COVID-19 severity or mortality individually but when analysed by haplotype, TC was associated with an increased risk of presenting critical COVID-19. Additionally, FokI CT genotype was more frequent in COVID-19 patients with hypertension, and T allele carriers presented higher aspartate aminotransferase levels. Our results suggest a relationship between VDR FokI and TaqI SNPs and COVID-19 severity in Mexican population. Although there are some previous reports of VDR polymorphisms in COVID-19, this represents the first report in Latin American population. Further studies on other populations are encouraged.

Genetic Diversity in Bronchial Asthma Susceptibility: Exploring the Role of Vitamin D Receptor Gene Polymorphisms in Varied Geographic Contexts.

Bronchial asthma (BA) exhibits varying prevalence across global populations, prompting a comprehensive investigation into genetic and environmental determinants. Vitamin D is a potent immunomodulator capable of suppressing inflammatory signals in several cell types involved in the asthmatic response; it exerts effects on the immune system by binding to the nuclear vitamin D receptor (VDR). VDR gene genetic variations are affecting serum vitamin D levels with a possible role in the BA risk. The current study aimed to examine the complex interaction of various factors (genetic background, serum vitamin D levels, and geographic location) to identify differences in the influence of these factors on the susceptibility to asthma between populations at different latitudes. Focusing on Eastern European cohorts from Latvia and Lithuania and comparing them with published data on East Asian populations, we explore the impact of VDR gene polymorphisms on BA susceptibility. Genotyping four key VDR SNPs and assessing their association with 25-hydroxyvitamin D levels, our study unveils significant associations of the studied loci with the risk of asthma-both risk-reducing and increasing effects, differently distributed between Baltic and East Asian populations. The functional effects of in silico VDR gene genetic variations are also identified and discussed.

Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Association between vitamin D receptor gene variants and neonatal sepsis: A systematic review and meta-analysis.

The objective of this systematic review and meta-analysis was elucidating the association of VDR SNPs (FokI, TaqI, BsmI, BgII, and ApaI) with neonatal sepsis. Literature search was performed to retrieve records published until August 2nd, 2023 (PROSPERO registration: CRD42023451355). Meta-analysis was carried out to determine the pooled estimates for Odds Ratio (OR). A total of four studies were included with 500 neonates (250 sepsis cases and 250 healthy controls). There was an association observed between TaqI SNP with neonatal sepsis for CT vs. CC+TT (OR=1.95) and TT vs CT+CC (OR=0.40). Moreover, the pooled estimates also suggested that CC vs. CT+TT (OR= 0.37) and C vs. T (OR=0.66) of FokI SNP were significantly associated with neonatal sepsis. SNP of BgII was found to be significantly associated with neonatal sepsis, but only reported in a single study.

Association between vitamin D receptor gene FokI polymorphism and mortality in patients with sepsis.

BACKGROUND: Sepsis is life-threatening organ dysfunction as a result of the host's dysregulated immune response to infection. The vitamin D receptor (VDR) gene FokI polymorphism influences immune cell behavior. In the present study, we aimed to investigate the association between VDR FokI polymorphism and mortality in sepsis and non-sepsis patients in the intensive care unit (ICU). METHODS AND RESULTS: This is a prospective observational study involving 96 sepsis and 96 non-sepsis patients admitted to the Ege University ICU. VDR FokI polymorphisms were investigated, as well as the relationship between the identified polymorphisms and mortality.  In-hospital mortality was 27.1% in the sepsis group and 8.33% in the non-sepsis group (p = 0.001). The frequencies of VDR FokI TT, TC, and CC genotypes were 8 (8.33%), 48 (50.0%), and 40 (41.7%) in the sepsis group, and 11 (11.5%), 42 (43.8%), and 43 (44.8%) in the non-sepsis group, respectively (p = 0.612). In the sepsis group, the frequencies of Fokl TT, TC, and CC genotypes did not differ significantly between survivors and non-survivors. However, homozygous C allele carriers had lower overall mortality (p = 0.047). CONCLUSION: The VDR FokI polymorphism, particularly the CC genotype, appears to be associated with lower mortality in ICU patients.

Molecular Mechanism of Vitamin D Receptor Modulating Wnt/ß-catenin Signaling Pathway in Gastric Cancer.

Background: Gastric cancer is the most common gastrointestinal cancer worldwide. The latest data showed that it was the fourth leading cause of cancer-related death. The unobvious symptom and the difficulties lying in the early diagnosis largely affect the effect of the treatment. Therefore, it becomes particularly important to investigate the related genes and signal transduction pathways in gastric cancer. Our previous study found that the vitamin D receptor (VDR) gene FokI polymorphism may be associated with susceptibility to gastric cancer in the Chinese Han population. However, the mechanism of VDR affecting gastric cancer is unknown. In this study, we explored the molecular mechanism and the possible signaling pathway of VDR modulating carcinogenesis and progression of gastric cancer. Methods: The expression of VDR in gastric cancer cell lines was interfered by plasmid transfection and RNA interference technology. And then we analyzed the cell viability and invasive ability by MTT assay, colony formation assay, and transwell migration assay, and detected the expression of VDR and several signaling proteins in gastric cancer cells by SDS-PAGE and Western blotting. Results: The overexpression of VDR can significantly inhibit the viability and invasive ability of gastric cancer cells; on the contrary, when VDR siRNA inhibits the expression of VDR, the viability and invasive ability of gastric cancer cells enhanced. VDR expression levels in gastric cancer cells treated with 1,25 (OH) 2D3 showed a time-dependent increased expression; and with the increase of the VDR expression, the expression of ß-catenin decreased gradually, but the expression of E-cadherin showed a time-dependent increase (P < 0.05). Compared with the mutant-type VDR gene(ff) cells, the degree of ß-catenin decline was significantly enhanced after transfected with homozygous wild-type VDR gene (FF) plasmids (p<0.05). Conclusions: The results of this study indicate that VDR FokI polymorphism plays an important role in the malignant phenotype of gastric cancer cells, such as proliferation, invasion, and clone formation. When the VDR is activated by its ligand, it can prevent the nuclear import of ß-catenin, affect the E-cadherin level, inhibit the proliferation of gastric cancer cells, which suggested that VDR FokI gene may play a role of cancer suppressor via Wnt/ß-catenin signaling pathway.

The CDX2 G allele and the FoKI F allele of the VDR gene are more prevalent and related to changes in vitamin D levels in patients with psoriasis vulgaris: A pilot study.

BACKGROUND AND AIMS: Psoriasis is a chronic, non-contagious autoimmune condition marked by dry, itchy,erythematous and scaly plaques. From modest, localized plaques to total body coverage, the severity of psoriasis varies. Plaque, guttate, inverted, pustular, and erythrodermic psoriasis are the five primary kinds. About 90% of cases are of plaque psoriasis, commonly known as psoriasis vulgaris. Study aims to determine the impact of an rs2228570 (FokI) variant and an rs11568820 (CDX2) variant on serum vitamin D levels (SVD) in patients with psoriasis, and the correlation between the two variants and disease severity. METHODS: A case-control study consisting of 95 psoriasis vulgaris patients and 84 healthy controls. The clinical investigation, molecular genetics analysis, and biochemical analysis were done for both groups. RESULTS: SVD levels were significantly decreased in psoriasis patients group. FokI genotypes analysis, we found no significant variance between groups. CDX2 G/G genotype is more prevalent in patients than controls. Moderate psoriasis vulgaris patients with CDX2 G/G genotypes have higher SVD levels than CDX2 G/A, and CDX2 A/A p = 0.003. CONCLUSION: The study found a difference in vitamin D levels between patients and healthy subjects, as well as a difference in vitamin D levels with different FoKI and CDX2 genotypes.

A Case-Control Study of the Fokl Polymorphism of the Vitamin D Receptor Gene in Bulgarians With Lumbar Disc Herniation.

BACKGROUND: The present study investigates whether vitamin D receptor (VDR) gene polymorphisms play a role in intervertebral disc degeneration (IDD), a common cause of low back pain (LBP) and reduced quality of life. Specifically, we examined the FokI VDR polymorphism and its potential association with lumbar disc herniation (LDH) in patients from Bulgaria. Previous studies have suggested a link between mutations in the VDR gene and IDD. METHODS: We investigated whether a potential connection between VDR polymorphisms and LDH was present by comparing the FokI polymorphism of 60 selected patients (25 to 60) with LDH and 60 healthy volunteers within the same age range. We used polymerase chain reaction to assess the phenotype of the examined subjects and statistical tests to evaluate whether the obtained results were statistically significant. RESULTS: The performed genetic and statistical analyses reviewed significant differences in genotypic frequencies between the patient group and healthy volunteers. The frequency of the F allele is notably higher in patients with LDH (80%) compared to volunteers (52%), while the f allele is more common among patients (86.6%) than volunteers (100%). CONCLUSION: This study strongly suggests that expression of the F allele of the VDR gene may increase the susceptibility of developing LDH, while having the f allele could potentially have a protective effect. Our results shed light on the underlying complex mechanisms contributing to the development of LDH.

Relevance of Serum Levels and Functional Genetic Variants in Vitamin D Receptor Gene among Saudi Women with Gestational Diabetes Mellitus.

Background: This study explored the association between ApaI-TaqI Single Nucleotide Polymorphisms (SNPs) in a Vitamin D receptor (VDR) and the risk of Gestational Diabetes Mellitus (GDM) in Saudi women, along with the serum levels of vitamin D. Methods: Ninety women with GDM and 90 non-GDM women were enrolled, based on the inclusion and exclusion criteria for pregnant women enrolled in a single-center study. Blood samples were retrieved from 180 pregnant women using ethylenediaminetetraacetic acid (EDTA) tubes. Serum samples were used to measure the vitamin D, 25-hydroxyvitamin D (25(OH)D or calcidiol), and lipid profiles. Blood was used to measure the hemoglobin A1c levels and to isolate the DNA. The polymerase chain reaction (PCR) was performed for the ApaI (rs79785232), BsmI (rs1544410), FokI (rs2228570), and TaqI (rs731236) SNPs in the VDR gene using restriction fragment length polymorphism analysis. Validation was performed using Sanger sequencing. Statistical analyses were performed between the patients with and without GDM using various statistical software packages. Results: The Hardy-Weinberg equilibrium analysis was statistically significant (p > 0.05). The ApaI, BsmI, and TaqI SNPs were associated with alleles, genotypes, and different genetic models (p < 0.05). Vitamin D levels were associated with deficient levels (p = 0.0002), as well as with a normal and overweight body mass index (p = 0.0004). When vitamin D levels were measured with GDM covariates, the fasting plasma glucose (FPG) (p = 0.0001), postprandial blood glucose (PPBG) (p < 0.0001), oral glucose tolerance test (OGTT)-1 h (p = 0.005), high-density lipoprotein (p = 0.022), and low-density lipoprotein cholesterol (LDLc) (p = 0.001) levels were significantly different. When similar vitamin D levels were measured for each genotype, we confirmed that the ApaI SNP was associated with sufficient levels (p < 0.0001), whereas the BsmI, FokI, and TaqI (p < 0.05) were associated with insufficient levels. The logistic regression model confirmed that the first hour of the OGTT (p = 0.005) was strongly associated with GDM, whereas the analysis of variance confirmed that FPG and PPBG (p < 0.05) were strongly associated with all the SNPs evaluated in the VDR gene. Additionally, the second hour of the OGTT (p = 0.048) and LDLc (p = 0.049) were associated with the ApaI and FokI SNP. Moreover, the first hour OGTT (p = 0.045) and lipid profile parameters (p < 0.05) were associated. Haplotype analysis revealed positive associations among the examined SNPs, which seemed compatible with the hypothesis that variants and combinations of multiple SNP genotypes enhance the risk of GDM in women. Haplotype analysis revealed that different combinations of alleles, such as AGCC, CATT, CGTC, AGTC, and CATT (p < 0.05), were strongly associated. The linkage disequilibrium (LD) analysis showed a strong association with all combinations (p < 0.05). Among the gene-gene interactions, all possible combinations showed a positive association (p < 0.05). Conclusions: Low vitamin D levels were observed in women with GDM. The ApaI, BsmI, and TaqI SNPs were associated with genotype and allele frequencies (p < 0.05). Vitamin D and the SNPs in the VDR gene were associated, according to the ANOVA, logistic regression, haplotype analysis, LD analysis, and the generalized multifactor dimensionality reduction model (p < 0.05).

Some genetic differences in patients with rheumatoid arthritis.

OBJECTIVE: Vitamin D is important for bone and cartilage metabolism. Changes in vitamin D blood level may be related to pathological disorders such as rheumatoid arthritis (RA). The main aim of this study is to investigate the association between RA and the vitamin D receptor (VDR) genes FokI and TaqI polymorphisms. One hundred RA patients and fifty healthy matched controls were assessed for VDR FokI and TaqI genotyping. Intact parathyroid hormone (PTH) and calcium (Ca) levels were measured, categorized, and compared between the cases and control groups. RESULTS: We found that the FokI genotype frequencies for the RA cases and control groups were FF:Ff:ff = 46%:52%:2% and 50%:50%:0%, respectively (P = 0.76). The TaqI genotype frequencies for the RA cases and control groups were TT:Tt:tt = 45%:44%:11% and 42%:42%:16%, respectively (P = 0.69). A statistically significant high serum PTH level was associated with the ff genotype (p = 0.03), and a significantly low serum Ca level was associated with the TT genotype (p = 0.003). In comparison with controls, no influence of VDR FokI and TaqI genotypes on RA susceptibility or risk was demonstrated.

Genetic Variability in Vitamin D Receptor and Migraine Susceptibility: A Southeastern European Case-Control Study.

Migraine is a common primary headache disorder with both environmental and genetic inputs. Cumulative evidence indicates an association between vitamin D and headache. Unravelling the precise role of vitamin D and its receptor in the pathophysiology of migraine can eventually contribute to more efficient prevention and management of this headache disorder. The aim of the study was to investigate the relation of the three most studied VDR variants, i.e., FokI (rs2228570), TaqI (rs731236) and BsmI (rs1544410), with migraine susceptibility and distinct clinical phenotypes in a Southeastern European case-control population residing in Greece. DNA was extracted from 191 unrelated patients diagnosed with migraine and 265 headache-free controls and genotyped using real-time PCR (LightSNiP assays) followed by melting curve analysis. Genotype frequency distribution analysis of the TaqI and BsmI variants showed a statistically significant difference between migraine cases and controls. In addition, subgroup analyses revealed a significant association between all three studied VDR variants, particularly with a migraine without aura subtype. Therefore, the current study provides supporting evidence for a possible association of VDR variants with migraines, particularly migraine without aura susceptibility in Southeastern Europeans residing in Greece, further reinforcing the emerging role of vitamin D and its receptor in migraines.

Increased susceptibility to complicated pneumonia among egyptian children with FokI (rs2228570), not TaqI (rs731236), vitamin D receptor gene polymorphism in association with vitamin D deficiency: a case-control study.

BACKGROUND: Determining a genetic contribution to the development of complicated community-acquired pneumonia in children may help understand underlying pathogenesis. We aimed to investigate the association between two vitamin D receptor (VDR) gene polymorphisms, FokI and TaqI, and susceptibility to complicated pneumonia in Egyptian children compared to uncomplicated pneumonia. Associations with 25 hydroxy-vitamin D serum level were studied. METHODS: This was a case-control study that included 320 participants divided into 2 groups: patients and controls. The patients' group included 100 children hospitalized with complicated pneumonia and 100 with uncomplicated pneumonia. 120 age and sex-matched apparently healthy children served as controls. The VDR FokI and TaqI polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. 25 hydroxy-vitamin D level was estimated in serum using ELISA. RESULTS: Regarding FokI, homozygous CC genotype was more common in complicated (52%) than uncomplicated pneumonia (28%) and controls (10%) (OR = 65; 95%CI (5.13-822.63), p < 0.001) and (OR = 4.3; 95%CI (0.7-27.16), p = 0.003), respectively. Children carrying C allele possessed 3 higher odds for complicated than uncomplicated pneumonia (OR = 3.08; 95%CI (1.33-7.14), p < 0.001). Heterozygous CT genotype increased susceptibility to complicated pneumonia (OR = 13.7; 95%CI (4.6-40.1), p < 0.001), not uncomplicated pneumonia (OR = 1.56; 95%CI (0.86-2.85), p = 0.145). Among complicated pneumonia, vitamin D level was lower in CC (6.92 ± 2.6ng/ml) than CT (9.55 ± 3.2 ng/ml) and TT genotype carriers (13.13 ± 3.6ng/ml) (p < 0.001). There was no significant difference between patients and controls as regards TaqI genotypes and alleles. CONCLUSION: In association with vitamin D deficiency, VDR gene FokI polymorphism, not TaqI, is a genetic risk factor for complicated pneumonia in Egyptian children.

Genetic susceptibility of vitamin D receptor gene polymorphisms on autosomal recessive primary microcephaly patients in Pakistani population: a case-control and in-silico study.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a rare genetic disorder that leads to reduced cerebral cortex caused by a mutation in corticogenesis. The expression of the Vitamin D receptor (VDR) gene is involved in the proliferation and differentiation of neural stem cells, and VDR polymorphisms have been associated with various neurological disorders. However, their relationship with MCPH has not been explored. This study aimed to investigate the association of VDR polymorphisms with MCPH due to its role in Wnt signaling pathway and its In-silico analysis. METHODS: Blood samples of 64 MCPH patients and 52 controls were collected to genotype VDR SNPs (TaqI (rs731236), FokI (rs2228570) and BsmI (rs1544410). In-silico tools were also used to assess the effects of exonic SNPs on mRNA and protein structure and pathogenicity of exonic and intronic SNPs. RESULTS: The study found that serum 25-OH vitamin D3 levels were significantly different in MCPH patients and healthy controls (P = 0.000). The genetic analysis showed that VDR polymorphisms of FokI and BsmI were seven times more frequent in MCPH patients than in controls (P < 0.05) and the recessive model for TaqI and dominant model for BsmI polymorphisms were also associated with the pathogenesis of MCPH. In-silico analysis showed that the pathogenicity effects of rs2228570 and rs1544410 are neutral while rs731236 causes a silent mutation which has no effect on VDR protein. CONCLUSION: VDR polymorphisms of FokI and BsmI are associated with the risk of MCPH. These findings suggest that VDR polymorphisms play a role in MCPH, which could provide important insights for understanding the molecular mechanisms of the disease.

VDR Gene Single Nucleotide Polymorphisms and Autoimmunity: A Narrative Review.

The vitamin D/Vitamin D receptor (VDR) axis is crucial for human health as it regulates the expression of genes involved in different functions, including calcium homeostasis, energy metabolism, cell growth and differentiation, and immune responses. In particular, the vitamin D/VDR complex regulates genes of both innate and adaptive immunity. Autoimmune diseases are believed to arise from a genetic predisposition and the presence of triggers such as hormones and environmental factors. Among these, a role for Vitamin D and molecules correlated to its functions has been repeatedly suggested. Four single nucleotide polymorphisms (SNPs) of the VDR gene, ApaI, BsmI, TaqI, and FokI, in particular, have been associated with autoimmune disorders. The presence of particular VDR SNP alleles and genotypes, thus, was observed to modulate the likelihood of developing diverse autoimmune conditions, either increasing or reducing it. In this work, we will review the scientific literature suggesting a role for these different factors in the pathogenesis of autoimmune conditions and summarize evidence indicating a possible VDR SNP involvement in the onset of these diseases. A better understanding of the role of the molecular mechanisms linking Vitamin D/VDR and autoimmunity might be extremely useful in designing novel therapeutic avenues for these disorders.

Role of FokI rs2228570 and Tru9I rs757343 Polymorphisms in the Mortality of Patients Infected with Different Variants of SARS-CoV-2.

BACKGROUND AND AIM: Low vitamin D levels are associated with the severity of coronavirus disease 19 (COVID-19). Vitamin D receptor gene polymorphisms, such as Tru9I rs757343 and FokI rs2228570, have been suggested to be potential risk factors for severe COVID-19 outcomes. This study investigated how Tru9I rs757343 and FokI rs2228570 polymorphisms influenced the mortality rate of COVID-19 in relation to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. METHODS: The polymerase chain reaction-restriction fragment length polymorphism assay was used to genotype Tru9I rs757343 and FokI rs2228570 genotypes in 1,734 recovered and 1,450 deceased patients. RESULTS: Our results demonstrated that the high mortality rate was correlated with FokI rs2228570 TT genotype in all three variants but was much higher in the Omicron BA.5 variant than in the Alpha and Delta variants. Furthermore, in patients infected with the Delta variant, FokI rs2228570 CT genotype was more highly correlated with the mortality rate compared to other variants. Thus, a high mortality rate was correlated with the Tru9I rs757343 AA genotype in the Omicron BA.5 variant, whereas this relationship was not observed in the other two variants. The T-A haplotype was related to COVID-19 mortality in all three variants, but its effect was more pronounced in the Alpha variant. Moreover, the T-G haplotype was significantly associated with all three variants. CONCLUSION: Our findings showed that the effects of Tru9I rs757343 and FokI rs2228570 polymorphisms were related to SARS-CoV-2 variants. However, further studies are still required to validate our findings.

A Pilot Study of the Association VDR Polymorphisms With Primary Hyperparathyroidism.

BACKGROUND/AIM: Primary hyperparathyroidism (PHPT) is the third most common endocrine disorder characterized by autonomous parathyroid hormone (PTH) production from one or more parathyroid glands and hypocalcemia. Vitamin D through its receptor is a principal regulator of parathyroid glands function. VDR gene polymorphisms, which affect the expression or structure of VDR protein, may be involved in the genetic pathogenesis of PHPT. The aim of this study was to investigate the role of FokI, ApaI, TaqI, and BsmI VDR gene polymorphisms as genetic predisposing factors for PHPT. PATIENTS AND METHODS: Fifty unrelated patients with sporadic PHPT and an equal number of corresponding ethnicity, sex and age range healthy volunteers were enrolled in the study. Genotyping was performed with polymerase chain reaction and restriction fragment length polymorphism assay. RESULTS: Statistically significant difference was observed in TaqI genotype distribution between PHPT patients and controls, while no association was detected for the other studied polymorphisms. CONCLUSION: TaqI TT and TC genotypes may be associated with PHPT risk in Greek population. Further independent studies are needed to replicate and validate the role of VDR TaqI polymorphism in PHPT predisposition.

FokI polymorphism of Vitamin D receptor gene and deficiency of serum Vitamin D increases the risk of breast cancer in North Indian women.

INTRODUCTION: Breast cancer is a leading cause of cancer morbidity and mortality in females. Decreased availability of Vitamin D within breast cells, contributed by deficiency of serum Vitamin D and polymorphisms of Vitamin D receptor genes are possible risk factors for breast cancer. OBJECTIVES: To study the association of FokI polymorphism of the Vitamin D Receptor gene with breast cancer in females and to study the levels of Vitamin D in breast cancer patients. MATERIALS AND METHODS: VDR gene FokI genotyping was done by PCR-RFLP method and levels of serum Vitamin D were estimated by ELISA. Statistical analysis was done with SPSS v.21. RESULTS: Serum Vitamin D was significantly lower in newly diagnosed breast cancer patients than in healthy controls (P = 0.016). Females with serum Vitamin D levels in the highest quartile have a lesser risk of breast cancer than those with serum Vitamin D levels in the lowest quartile (O. R = 2.4421, C.I = 1.09-5.45, P = 0.029). The risk of developing breast cancer is higher in women with the polymorphic T allele for VDR FokI genotype (CT/TT) than those homozygous for the wild C allele (CC). (O.R. = 4.295, C.I. = 2.2110-8.3451, p-value = <0.0001). Levels of serum Vitamin D were significantly higher (p < 0.001) in ER + patients and significantly low in those presenting with higher stages of cancer (p = 0.009). CONCLUSIONS: FokI polymorphism of VDR gene and low circulating Vitamin D levels increase the risk of developing breast cancer in North Indian females. Serum Vitamin D can be used as a prognostic factor.

Role of the Vitamin D Receptor (VDR) in the Pathogenesis of Osteoporosis: A Genetic, Epigenetic and Molecular Pilot Study.

The vitamin D receptor (VDR) regulates bone development and calcium homeostasis, suggesting a central role in musculoskeletal diseases such as osteoporosis (OP). Several studies have examined the contribution of VDR polymorphisms and epigenetic signatures in bone metabolism and OP risk, with sometimes inconclusive results. Our study aimed to explore the association between genetic variability, expression and the methylation pattern of VDR with the risk of OP in a cohort of Caucasian patients. Genomic DNA from 139 OP, 54 osteopenic (Ope) and 73 healthy (CTR) subjects were used for genotyping the rs731236 (TaqI), rs2228570 (FokI) and rs11568820 (Cdx2) polymorphisms of the VDR gene by an allelic discrimination assay. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of VDR expression levels and pyrosequencing analysis of a VDR promoter CpG island were carried out in a subcohort (25 OP and 25 CTR) of subjects. Data obtained showed a significantly higher OP risk for rs11568820 G/A and A/A genotypes (p = 0.05). qRT-PCR revealed lower VDR gene expression levels in the OP group compared to CTR subjects (p = 0.0009), also associated with both the rs11568820 A/A genotype (p = 0.03) and femoral fragility fractures (p = 0.05). No association was found between the methylation pattern of the region analyzed of the VDR promoter and its expression levels. Our results identify a significative association between Cdx2 rs11568820 polymorphism and OP risk. In addition, the VDR transcriptomic profile suggests a putative interconnection with OP progression, providing a useful tool to stratify OP phenotype and fragility fracture risk.