Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 31  |  Issue : 2  |  Page : 455--461

Vitamin D receptor gene polymorphism in immune thrombocytopenic purpura


Mohamed Abdelhafez1, Enas S Esaa2, Mohamed Sakr1,  
1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Correspondence Address:
Mohamed Sakr
6 El-Geish Street, Queissna, Menoufia
Egypt

Abstract

Objective The aim of this study was to assess the association of vitamin D receptor (VDR) gene polymorphism BsmI in cases of primary immune thrombocytopenic purpura (ITP). Background Recently, several studies have demonstrated the role of VDR polymorphisms in the development of autoimmune diseases. Vitamin D affects both innate and adaptive immune responses, which have been held responsible in ITP pathogenesis. Patient and methods VDR polymorphism BsmI (rs1544410) was detected by PCR followed by restriction fragment length polymorphism analysis. DNA samples were extracted from the peripheral blood of 40 ITP patients and 60 geographically and ethnically matched healthy controls. Results A statistically significant difference was found in the BsmI polymorphism between ITP patients and controls (χ2 = 8.77, P = 0.01). The BsmI polymorphism B allele was higher in ITP patients compared with controls but with a statistically insignificant difference (χ2 = 2.125, P = 0.145). The bb genotype played a protective role in ITP incidence. Conclusion This is the first published report on VDR gene polymorphisms in adult ITP patients. The BsmI genotype was associated with increased risk for ITP incidence with no obvious effect on bleeding severity, platelet count, or site of bleeding.



How to cite this article:
Abdelhafez M, Esaa ES, Sakr M. Vitamin D receptor gene polymorphism in immune thrombocytopenic purpura.Menoufia Med J 2018;31:455-461


How to cite this URL:
Abdelhafez M, Esaa ES, Sakr M. Vitamin D receptor gene polymorphism in immune thrombocytopenic purpura. Menoufia Med J [serial online] 2018 [cited 2024 Mar 28 ];31:455-461
Available from: http://www.mmj.eg.net/text.asp?2018/31/2/455/239753


Full Text



 Introduction



Vitamin D is a steroid hormone precursor that undergoes chemical conversion in the liver and kidney: the first reaction produces 25-hydroxyvitamin D, an objective indicator of vitamin D status, and the second produces the main bioactive form, 1,25-dihydroxyvitamin D [1]. Vitamin D has been shown to exert various anti-inflammatory and immune-modulatory effects, along with its major role in bone mineral homeostasis [1]. Vitamin D directly acts on immune cells by promoting monocyte differentiation and by inhibiting lymphocyte proliferation and production of immunoglobulins and cytokines, such as interleukin-2, interferon-γ, and interleukin-12 [1],[2]. It also inhibits dendritic cell differentiation and maturation [1], and reduces the expression of major histocompatibility complex class II molecules on both immune and nonimmune cells [1],[3]. Vitamin D inhibits T helper (Th) 1 and Th17 responses [4],[5], whereas it promotes the expressions of Th2 cytokines [6] and enhances the ability of T regulatory cells to suppress T-cell proliferation [7],[8]. As a consequence, both cell-mediated immune response and B cells proliferation and autoantibodies production would directly be downregulated by vitamin D [1],[2],[3]. Such actions result in an overall protective effect of vitamin D against immune-mediated diseases. In humans, vitamin D status has been associated with susceptibility to several immune-mediated disorders including chronic infections (tuberculosis) and autoimmune diseases [7],[8],[9],[10],[11],[12],[13],[14], and administration of vitamin D supplements has been reported to reduce the risk to develop such diseases [1],[10],[13],[15].

The pleiotropic effects of vitamin D are exerted via the vitamin D receptor (VDR), which belongs to the steroid receptor superfamily and is widely expressed in many cell types including lymphocytes, macrophages, and several endocrine cells [11]. The VDR gene, located on chromosome 12q12–q14, shows an extensive polymorphism that influences its function. Four major single nucleotide polymorphisms (SNPs) have been intensively studied – namely, FokI in exon 2, BsmI and ApaI in intron 8, and TaqI in exon 9 – clustered in several haplotype blocks of extensive linkage disequilibrium. BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236) SNPs are in strong linkage disequilibrium with each other, whereas no significant linkage disequilibrium with the FokI site was observed. The ApaI (G/T substitution), BsmI (A/G substitution), and TaqI (T/C substitution) polymorphisms do not produce any structural change on the VDR protein [11],[12]. On the other hand, the FokI (T/C substitution) polymorphism introduces a second start codon in the VDR gene and yields two potential initiation sites with actual structural change on the VDR protein, suggesting a potential functional consequence.

Certain SNPs of the VDR gene may result in reduced vitamin D function and have been associated with a range of autoimmune diseases such as systemic lupus erythematosus [16],[17], multiple sclerosis [18],[19], type 1 diabetes [20],[21], and Grave's [22], and Behcet's disease [23].

Concerning immune thrombocytopenia, there are no published reports available in the literature on the association of VDR gene polymorphisms in adult immune thrombocytopenic purpura (ITP) patients.

Primary ITP is a complex, chronic, often cell-specific, autoimmune disease that is still not fully understood. It is defined currently as isolated thrombocytopenia (peripheral blood platelet count 9/l) in the absence of conditions known to cause thrombocytopenia [24]. According to population-based studies, the overall incidence of ITP ranges from 3.2 to 12.1 per 105 adults each year, with prevalence ranges from 9.5 to 23.6 per 105 persons. The incidence of ITP increases with age [25]. The improved understanding of the innate and adaptive immune systems, however, allows us to understand and appreciate some of the complex interactions between platelets, the immune system, and the development of ITP. Immune-mediated platelet destruction and/or insufficient platelet production in ITP occurs by a complex process involving multiple components of the immune system [26].

The present study aimed to assess the association of VDR gene polymorphism BsmI in cases of primary ITP.

 Patients and Methods



This was a case–control study. It included 40, consecutive, primary, adult, female ITP cases diagnosed according to the guidelines of the American Society of Hematology 2011 [27]. Patients were enrolled during their admission to inpatient wards or routine follow-up at the hematology clinic, Menoufia University Hospitals, during the period from April 2014 to March 2016 inclusive. Sixty, unrelated, healthy controls matched for age and sex were included as a control group. Patients were invited to participate with no attempt to select them by known or perceived risk factors.

Informed required consents were obtained from the patients and controls in advance. All investigations were performed in accordance with the Menoufia University, Health and Human Ethical Clearance Committee guidelines for Clinical Research. The Local Ethics Committee approved the study protocol.

For all ITP patients and controls, we carried out full history taking, including family history; physical examination with emphasis on bleeding symptoms, severity, and site; abdominal ultrasound imaging; and laboratory investigations such as complete blood count with peripheral blood smear, direct antiglobulin test, virology screen, stool test for Helicobacter pylori antigen, antinuclear antibodies, and antiphospholipid antibodies, pregnancy test, and bone marrow examination when needed.

Research investigation

The VRD BsmI polymorphism (rs1544410) was detected by PCR followed by restriction fragment length polymorphism analysis.

Blood sampling

One milliliter peripheral venous blood samples were collected in sterile tubes containing EDTA for DNA extraction.

DNA extraction

DNA was extracted from whole-blood samples using QIA amp Blood Genomic DNA Kit that depends on the spin column method, according to the protocol supplied by the manufacturer (Qiagen GmbH, Hilden, Germany). For determining DNA concentration, 1 OD unit measured at 260 nm corresponds to 50 μg/ml of DNA. DNA purity was determined by measuring the A260/A280 ratio. The ratio of 1.8–1.9 corresponds to pure double-stranded DNA. DNA sample aliquots were stored at −20°C until use.

Analysis of polymorphisms

The analysis was performed using 1× Taq PCR master mix (Taq PCR Master Mix Kit; Qiagen GmbH), which contained 200 mol/l of each dNTP, 5ml of 109 reaction buffer, 1.25 U Taq gold polymerase, and 4 mmol/l MgCl2. For a 25 μl reaction volume, 12.5 μl master mix was used and then DNA and PCR primers (Promega, Madison, Wisconsin, USA) were added. The volume of the reaction mixture was increased to a final volume of 25 μl with the addition of dH2O. The BsmI PCR primers were as follows: forward: 5'-CAACCAAGACTACAAGTACCGC GTCAGTGA-3' and reverse: 5'-AACCAGCGG GAAGAGGTCAAGGG-3'. Amplification was performed using a PTC-100 thermal cycler (MJ Research Inc., Watertown, Massachusetts, USA) with 65°C as the annealing temperature. Restriction fragment length polymorphism was used to identify VDR genotypes. Amplified PCR products (10 ml) were digested (37°C for 20 h) with 4 U BsmI restriction enzyme (New England Biolabs, 75-77 Knowl Piece, Wilbury Way, Hitchin, Herts SG4 0TY) in 20 μl reaction volume; The EC 360 Submarine Gel electrophoresis system was used (Maxicell, EC 360; MEC Apparatus Cooperation, St Petersburg, Florida, USA). The PCR products were visualized using 2% agarose gel containing ethidium bromide under ultraviolet transillumination.

Capital letters represented absence and lowercase letters represented presence of a BsmI restriction site (B/b). Genotype was determined according to fragment length – that is, homozygote AA (bb) individuals = 822 bp product, heterozygote GA (Bb) individuals = 822, 650, and 172 bp products, and homozygote GG (BB) individuals = 650 and 172 bp products [Figure 4].{Figure 4}

A SNP resulting in A–G substitution in the VDR gene intron 8 led to the generation of a BsmI restriction site. Homozygous individuals with alleles containing nucleotide A at this position showed one band at 822 bp and were designated as having bb BsmI genotype. Homozygous individuals with alleles containing G at this position showed two bands of 650 and 172 bp and were designated as BB. Individuals with heterozygote status showed three bands 825, 650, and 172 bp and were designated as Bb. For quality control, genotyping of 10% of the samples was repeated and interpreted blindly by two different observers, which proved to be identical to the initial results.

The included patients in our study were divided in two groups – ITP cases and healthy controls. The ITP group was further divided according to bleeding severity to mild and moderate following (modified Buchanan and Adix and, Imbach 2013) [25],[26] and International working group for ITP [27].

Statistical procedure

Data were analyzed using statistical package for social sciences, version 18 (SPSS Inc., Chicago, Illinois, USA). Quantitative data are expressed as mean ± SD. Qualitative data are expressed as frequencies and percentages. For qualitative data, the c2-test with Yates correction or Fisher's exact test was used when appropriate. P value was significant if less than 0.05; strength of associations was assessed by computing odds ratio and their 95% confidence intervals. The Kolmogrov–Smirnov test was used to explore normality of data. The Student t-test and one-way analysis of variance test were used to compare between quantitative data of two and more than two groups, respectively.

Bonferroni's correction was used for the obtained P value by multiplying the observed (uncorrected) P value by the number of comparisons or tests.

The significance of association between the observed and expected number of the genotypes for a population in the Hardy–Weinberg equilibrium was analyzed using Pearson's two-sided c2-test.

 Results



Demographics and baseline characteristics of ITP patients are summarized in [Table 1] and [Table 2]. This study included 40, adult, female ITP patients (age range: 20–43 years, mean ± SD: 30.52 ± 5.66586) and 60, adult, female controls matched for age (range: 20–40 years, mean ± SD: 29.51 ± 5.84399). There was a highly statistically significant difference between the ITP group and the control group regarding hemoglobin level and platelet count.{Table 1}{Table 2}

In our study, there was a statistically significant difference between ITP patients and controls with regard to distribution of BsmI genotype frequencies (P = 0.01) with no statistically significant differences regarding the distribution of the BsmI genotype allele (P = 0.145). Moreover, there was no statistically significant differences between mutant and corecessive BsmI genotype variants in the ITP group and the control group (P = 0.15) and between codominant and recessive BsmI genotype variants (P = 0.08) [Table 3].{Table 3}

This study showed no significant differences between BsmI genotype and hemoglobin level (P = 0.69) and platelet count (P = 0.32) [Figure 1], [Figure 2], [Figure 3].{Figure 1}{Figure 2}{Figure 3}

Regarding bleeding severity, there was no statistically significant differences with respect to BsmI genotype frequency (P = 0.08) and allele (P = 0.206) [Table 4]. There was a statistically significant difference between BB versus Bb + bb genotype variants regarding degree of bleeding severity (P = 0.014) with no significant differences between BB + Bb versus bb genotype variants (P = 0.12) [Table 4].{Table 4}

For site of bleeding, the BsmI genotype and its variants showed no statistically significant differences apart from oral bleeding where BB + Bb is in statistical significant differences when compared with bb genotype (P = 0.04) and BB when compared with Bb + bb (P = 0.03) [Table 5], [Table 6], [Table 7].{Table 5}{Table 6}{Table 7}

 Discussion



As ITP had been testified to be a heterogeneous disease, studies in terms of gene polymorphisms in ITP are being carried out extensively, and new findings and opinions are continually being published,[28-30] which are helpful to better understand the pathogenesis of ITP and would be of value in choosing therapeutic regimens.

Several cells involved in the immune system express VDR and key vitamin D metabolizing enzymes, which could explain the suppressive effects of vitamin D on immunity [31].

Most of the biological activities of vitamin D are mediated by the VDR gene. Genetic variation in the VDR gene could lead to significant receptor dysfunction, which could affect calcium metabolism, cell proliferation, and immune response. Polymorphisms in the VDR gene have been associated with bad health outcomes involving low bone density, cardiovascular disease, cancers, autoimmunity, and infections. VDR polymorphisms have been reported to be associated with a wide range of autoimmune diseases (such as rheumatoid arthritis, systemic sclerosis, type 1 diabetes mellitus, multiple sclerosis, inflammatory bowel diseases, and autoimmune gastritis).

BsmI is located in the intron between exons 8 and 9, which may affect VDR mRNA stability. Since 1996, when Berg et al. first reported that the VDR BsmI polymorphism could affect osteoporosis in postmenopausal women, [32] to date, a large number of studies regarding the association between BsmI gene polymorphism and autoimmune diseases have been published.

Despite an extensive literature study, no study has so far reported the relationship between VDR polymorphisms and adult ITP. This is the first report on VDR gene BsmI polymorphisms in adult ITP.

In the present study, there was a statistically significant difference between the ITP group and the control group regarding BsmI genotype frequency with no statistically significant difference regarding the BsmI allele.

The heterozygous pattern Bb genotype was the most common among patients and least common among controls. The frequency of the B allele in the present study was higher, although insignificantly, in ITP patients than in controls. The b allele was higher, although insignificantly, in controls than in patients.

In our study, there was no statistically significant difference between BsmI genotype and its variants and clinical parameters of ITP presentations (platelet count, bleeding severity, site of bleeding) apart from oral bleeding where it was common in B+ ve allele and not in BB.

Among controls, the distribution of the BsmI genotype was BB, Bb, and bb at 33.3, 21.7, and 45.0%, respectively. The percentage of BB was similar to values obtained by Saad et al. [32] (Egypt), Abd-Allah et al. [33] (Egypt), and El-Beshbishy et al. [34] (Saudi Arabia), but in contrast to the results of Karray et al. [35] (Tunisia), Cai et al. [36] (China), and Carvalho et al. [37] (White). The percentage of Bb was similar to values obtained by Emerah and El-Shal [38] (Egypt), Mosaad et al. [39] (Egypt), and Khalid [40] (Sudan), but in contrast to the results of Abdeltif et al. [41] (Morocco), Abbasi et al. [42] (China), Sakulpipatsin et al. [43] (Taiwan), Mostowska et al. [44] (White), and Kizildag et al. [45] (Turkey). The percentage of bb was similar to the values obtained by Abd-Allah et al. [33], Mosaad et al. [39], and Khalid [40], but in contrast to the results of Karray et al. [35], Abdeltif et al. [41], Sakulpipatsin et al. [43], and Carvalho et al. [37].

These variations are due to ethnic variations in VDR polymorphisms among populations with gene–gene and gene–environment interactions.

Moreover, in the control group, the BsmI B allele frequency was 44.2%. This result is in agreement with El-Hoseiny et al. [46] but in contrast to the results of Emerah and El-Shal [38] and Mansour et al. [47]. The b allele frequency in the control group was 55.8%. This is in agreement with El-Barbary et al. [48] and Abd-Allah et al. [33] but not in agreement with Saad et al. [32] and Mosaad et al. [39]. These differences may be related the size of each study.

Finally, we can recommend that the VDR BsmI polymorphism can be used as a risk marker for primary ITP susceptibility; however, further studies on larger samples and replication of significant findings are necessary to clarify this notice. It is better to assess the levels of 25-hydroxyvitamin D while checking VDR polymorphisms and to take into account all polymorphisms that could influence the expression and activity of the mRNA and that in high linkage disequilibrium with BsmI (ApaI, TaqI polymorphisms). Close follow-up of ITP patients during treatment is highly indicated to study the drawbacks or advantages of BsmI genotype on treatment plan.

 Conclusion



In our study, we found an association of VDR BsmI in ITP patients with no obvious effect on platelet count, bleeding severity, or even site of bleeding.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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