Gene detection of VDR BsmI locus and its approteins, genes and growthplication in rational drug use in patients with osteoporosis

Abstract

Aim: This paper determines the polymorphism distribution of the VDR BsmI gene in 350 patients and provides medication recommendations for osteoporosis based on detection results. Materials & methods: Chi-square tests compared genotype and allele frequencies with other populations. Results: Genotype frequencies were 91.66 bb, 8.72 Bb and 0.21% BB, with allelic frequencies of 95.43 b and 4.57% B, adhering to Hardy–Weinberg equilibrium. These findings suggest that VDR gene polymorphisms, particularly at the BsmIlocus, play an essential role in bone health and osteoporosis treatment. Genotype-based drug selection reduced adverse reactions from 14 to two cases. Conclusion: These findings improve clinical treatment efficacy and guide rational drug use for osteoporosis patients.

Article highlights

Objective

• To determine the polymorphism distribution of the vitamin D receptor (VDR) BsmI locus gene in osteoporosis patients and provide medication recommendations.

Methods

• Genetic testing of 350 patients for VDR BsmI gene polymorphisms, comparison of genotype and allele frequencies, and assessment of the impact on drug selection.

Significance

• Understanding the role of VDR gene polymorphisms in bone health and treatment response, and the potential for genotype-based drug selection.

Results

• Genotype distribution: The study identified three genotypes at the BsmI locus: bb (91.66%), Bb (8.72%) and BB (0.21%), with allele frequencies of 95.43% for b and 4.57% for B. This distribution aligns with the Hardy–Weinberg equilibrium, indicating a representative sample.

• Comparison with other populations: The VDR gene allele frequencies in the Guangdong Han population significantly differ from those in South Asia, America, Europe and Africa but are similar to other East Asian populations.

• Impact on bone mineral density: There was no significant difference in bone mineral density T-scores before and after treatment across the different VDR genotypes, but patients with the b allele showed better responses to osteoporosis treatments, particularly with bisphosphonates.

• Adverse drug reactions (ADRs): before genetic testing, there were 14 ADRs, which reduced to only two cases post implementation, indicating improved treatment outcomes and reduced side effects through genotype-based drug selection.

Conclusions

• The study supports the importance of genetic testing in guiding osteoporosis treatment as VDR gene polymorphisms, particularly at the BsmI locus, significantly influence treatment efficacy and the occurrence of ADRs.

• Genotype-based drug selection can improve clinical outcomes and minimize side effects.

• The findings suggest the need for further research on the distribution and impact of VDR BsmI locus polymorphism in different Chinese populations. Large-scale, multicenter studies are recommended to validate these results and enhance the precision of genotype-based drug recommendations.

• Integrating pharmacogenomics into clinical practice promises to improve the effectiveness of osteoporosis treatments, minimize adverse reactions, and advance personalized medicine, ultimately enhancing patient outcomes and quality of life.

Keywords: :

• genetic polymorphism
• genetic testing
• genotype
• osteoporosis
• vitamin D receptor (VDR) gene

1. Background

The White Paper on Prevention and Treatment of Osteoporosis in China [1] highlights the growing concern of an aging population in our country. It reports that approximately 70 million individuals aged over 50 are afflicted with osteoporosis, and about 210 million are affected by low bone mass. As the situation deteriorates, osteoporosis has become a prominent public health issue. Osteoporosis, a chronic bone disorder, is characterized by compromised bone strength, an increased risk of fractures and a strong association with aging. Essential elements for normal bone growth and structural integrity, such as vitamin D and calcium, are critical in maintaining bone health [2]. A deficiency in these nutrients is linked to osteopenia and osteoporosis. Therefore, the clinical selection of drug therapy emerges as an effective strategy in combating osteoporosis.

Osteoporosis is a multifactorial disease influenced by complex mechanisms, encompassing both genetic and environmental factors. Among these, genetic factors primarily affect the bone size, bone mass, structure, microstructure and internal characteristics, with 60–80% of peak bone mass attributable to genetic determinants [3]. Furthermore, extensive studies, including twins and family, have firmly established the pivotal role of genetic factors in bone mass regulation [4]. The vitamin D receptor (VDR) gene, encoding the vitamin D nuclear hormone receptor, is extensively expressed in various tissues and cells. It plays a critical role in regulating calcium and phosphorus metabolism and bone mineralization [3]. Functional mutations in this gene are crucial for the balance of bone metabolism. Study demonstrating the link between VDR gene single nucleotide polymorphisms (SNPs) and bone mineral density (BMD) [5], extensive research has been conducted globally. Common polymorphic sites within the VDR gene include BsmI, ApaI, FokI and TaqI. Research indicates that polymorphisms at the VDR gene’s BsmI locus are associated with the development of osteoporosis [6]. Such mutations occur at a detectable frequency within the populations, influencing gene transcription and protein expression. The Bsml and Apal restriction sites are located in intron 8, Taq1 in intron 9 and Fokl at the 5′ end of the gene. The expression of VDR gene polymorphisms varies significantly across different populations and individuals, with prevalences as high as 0.1%, termed SNP [7]. At the BsmI locus, three possible genotypes can occur: wild-type (bb), heterozygous mutation (Bb) and homozygous mutation (BB).

Several studies have demonstrated that variations in the genotypes at the BsmI locus of the VDR gene can influence the receptor’s functionality, thereby impacting the metabolism and activity of vitamin D. Notably, mutations at this locus, including both heterozygous and homozygous forms, have been associated with a reduced activity of the VDR, potentially increasing the risk of diseases such as osteoporosis and breast cancer. However, research on the factors influencing the distribution of the BsmI locus in the VDR gene within the Chinese population remains limited, indicating a need for further investigation into this locus’s distribution.

In this study, we aim to further explore the distribution of polymorphisms at the BsmI locus of the VDR gene among the Han population in Guangdong Province and to examine the relationship between the mutation rate at this locus and the incidence of osteoporosis.

2. Research methodology

2.1. General information

A total of 350 people were selected who underwent bone examination in the Department of Surgery and Internal Medicine, aged 17–95 years, with an average age of 63.77 years and a median age of 65.5 age, and those with genetic or other body abnormalities were excluded.

2.2. Main instruments & reagents

Multichannel fluorescence quantitative analyzer (Fascan 48S Xi'an Tianlong Technology Co., Ltd); Vortex mixer (MIX1000 Shanghai Jingsheng Scientific Instrument Co., Ltd); Metal bath (DH300 Hangzhou Ruicheng Instrument Co., Ltd); Pocket centrifuge (MC6000 Hangzhou Union Instrument Co., Ltd); adjustable micropipette (20–200 ul, 2–20 ul, 0.5–10 ul Eppendorf, Germany); desktop high-speed centrifuge (Beckman, USA); gene detection reagents, nucleic acid extraction reagents (Guangzhou Hisilicon Medical Technology Co., Ltd).

2.3. Methods

2.3.1. Specimen collection

At the initial visit, 2–3 ml of peripheral venous blood was collected from each participant using ethylenediaminetetraacetic acid (EDTA-K2) as an anticoagulant. The samples were stored at 4–8°C and processed within 1 week.

2.3.2. Genetic testing

Whole blood samples were analyzed using a multichannel fluorescence detector to identify SNPs at the VDR gene BsmI (rs154410) locus, following the kit’s instructions. The results were digitally processed and recorded.

2.4. Statistical processing

Data analysis were performed using SPSS 25.0 statistical software. The Hardy–Weinberg equilibrium test was applied to assess the representativeness of the sample population. Chi-square tests were used to compare the frequency of VDR gene BsmI (rs154410) locus genotyping and allele frequencies between groups. With p < 0.05 considered statistically significant.

3. Results

3.1. Distribution characteristics of VDR gene BsmI polymorphism

In this study, the BsmI polymorphism of the VDR gene was analyzed in 350 individuals from Foshan, Guangdong. The genotype distribution frequencies were found to be 91.66% for wild-type homozygotes (bb), 8.72% for Bb and 0.21% for BB. The allele frequencies was 95.43% for the b allele and 4.57% for the B allele. The results conformed to the Hardy–Weinberg equilibrium law (χ² = 0.057; p = 0.9719), indicating that the sample population was representative.

Furthermore, among males in Foshan, Guangdong, the frequencies of the VDR genes genotypes bb, Bb and BB were 88.15, 11.85 and 0.74% respectively. In the female population, the frequencies of the VDR gene alleles b and B were 94.07 and 6.67%, respectively. The genotype frequencies for bb and Bb were 93.46 and 6.54%, respectively, while the allele frequencies of b and B were 96.73 and 3.27%, respectively. No significant difference were observed in allele or genotype frequencies between the male and female populations (p > 0.05). Additionally, no significant differences in distribution were noted when compared with the reported results from the Han population in other regions such as Shanghai, Fuzhou, Harbin, Xinjiang, Guangzhou (p > 0.05), with the bb genotype being predominantly observed, ranging from 83.6 to 91.66%. Refer to Tables 1 & 2 for detailed distributions.

Table 1. Frequency distribution of vitamin D receptor genotypes in male and female populations in Guangdong [n (%)].

Parameters			VDR Genotype				Frequency
Sex	Sum	bb	Bb	BB	p-value^2	p-value	b	B	p-value^2	p-value
M	136	119 (88.15)	16 (11.85)	1 (0.74)	4.578	0.101	127 (94.07)	9 (6.67)	3.698	0.157
F	214	199 (93.46)	14 (6.54)	0 (0.0)			207 (96.73)	7 (3.27)

F: Female; M: Male; VDR: Vitamin D receptor.

Table 2. Comparison of vitamin D receptor genotype frequencies in this region and the country and other regions (n [%]).

Region	Sum (n)	VDR Genotype					Frequency
		bb	Bb	BB	χ^2	p-value	b	B	χ^2	p-value
Foshan (study)	350	319 (91.66)	30 (8.72)	1 (0.21)			324 (95.43)	16 (4.57)
Guangzhou	368	325 (88.3)	42 (11.4)	1 (0.3)	0.434	0.804	692 (94.0)	44 (6.0)	0.204	0.651
Xinjiang	336	290 (86.3)	36 (10.7)	10 (3)	2.787	0.248	616 (91.7)	56 (8.3)	1.155	0.282
Harbin	98	82 (83.7)	16 (16.3)	0	2.866	0.238	90 (91.8)	8 (8.2)	1.102	0.293
Fuzhou	150	134 (89.4)	11 (7.3)	5 (3.3)	2.873	0.237	139.5|(93)	10.5 (7)	0.542	0.461
Shanghai	611	511 (83.6)	100 (16.4)	0	2.927	0.231	561 (91.8)	50 (8.2)	1.102	0.293

VDR: Vitamin D receptor.

3.2. Comparison of allele frequencies in different populations in 1000 Genomes Database

The 1000 Genomes database (www.internationalgenome.org/) revealed significant differences in the distribution of VDR gene allele frequencies between the Han population in Guangdong and populations in South Asia, America, Europe and Africa (p < 0.05). In contrast, comparisons with East Asian populations showed no statistical differences in allele distributions (p > 0.05), refer to Table 3.

Table 3. Comparison of allele frequencies of different populations with the Thousand Genomes Database (n [%]).

	Allele	Guangdong	East Asia	South Asia	America(a)	Europe(a)	Africa(a)
Point		n = 350	n = 1008	n = 978	n = 694	n = 1006	n = 1322
VDR	b	95.42	93.6	51.5	74.4	59.6	72.8
	B	4.57	6.4	48.5	25.6	40.4	27.2

Note: a compared with Guangdong, p < 0.05.

VDR: Vitamin D receptor.

3.3. Comparison of BMD T scores of the same people before & after genetic testing

Following the initiation of a genetic testing project in our hospital, data from 350 patients were analyzed, and drug recommendation reports based on genetic testing outcomes were utilized clinically. Clinicians prescribed treatments aligned with these recommendations. Medical records indicated that the patients experienced notable improvements in treatment outcomes and significant enhancements in related health indicators. A comparative analysis of BMD T-scores for these patients before and after genetic testing revealed the following results, detailed in Table 4.

Table 4. Comparison of bone mineral density T scores before and after treatment.

Bone Region	Number of cases	BMD before treatment	BMD after treatment
Lumbar vertebrae 2–4 (x ± s)	18	-3.98 ± 1.03	-3.87 ± 1.16

BMD: Bone mineral density.

3.4. Results of adverse reactions before & after genetic testing

During the period from 2018 and 2020 at our hospital, clinicians prescribed medications to osteoporosis patients based solely on their experience. As a result, 14 adverse reactions during the treatment period. However, following the implementation of genetic testing in 2021, the pharmacy department been providing clinicans with drug recommendations based on genetic test results. which resulted in only two cases of adverse reactions.

4. Discussion

Vitamin D plays a critical role in the normal metabolism of bone, operating through VDR that are ubiquitously distributed across somatic cells. The VDR gene is located on the long arm of human chromosome 12 (12q13.11) and consists of 9 exons and 8 introns. Key polymorphisms have been identified, including BsmI (rs1544410) in the 8th intron, ApaI (rs7975232), TaqI (rs731236) in exon 9 and FokI (rs2228570) at the 5′ end [8]. Study has demonstrated a correlated between VDR gene polymorphisms and the absorption of vitamin D [9]. Specifically, healthy girls carrying the b allele at the BsmI locus exhibited a significantly higher percentage of BMC after vitamin D supplementation compared with those with the wild-type (BB) genotype. Additionally, the percentage change in BMC in the Bb genotype group was significant different from that in the placebo group (p < 0.05).

4.1. VDR gene polymorphism (BsmI) distribution

The results of this study revealed that in the Han population in Foshan, the distribution of BsmI genotypes was as follows: BB at 0.21, Bb at 8.72 and bb at 91.66%. This frequency distribution is consistent with findings from other regions of China, such as Guangzhou and Shanghai. Reports suggest that the proportion of bb genotypes in East Asian populations is typically high, generally around 90% [10]. In contrast, the distribution of VDR genotypes among Xinjiang Uygur, Kazakh and Mongolian populations displays significant differences (p < 0.001). These patterns resemble those observed in Caucasian population [11], where the proportion of the Bb genotype is notably high, reaching about 50%. This highlights significant ethnic variations in the frequency distribution of VDR genotype polymorphisms.

4.2. VDR gene polymorphism (BsmI) distribution & BMD

Our findings indicated no significant differences in genotype frequencies between males and females, nor between our study population and those in other regions such as Shanghai, Fuzhou, Harbin, Xinjiang and Guangzhou. Multiple SNPs in the VDR gene have been identified, which are believed to influence bone metabolism significantly. However, results vary substantially across different regions and ethnic groups. The association of the VDR gene with osteoporosis remains controversial [12]. Meta-analyses on the relationship between BsmI loci and BMD have also had inconsistent reports [13–16]. Such disparities can be attributed to a range of factors, including age, race, geographic location, sample size and methodology. Environmental factors, such as nutrition, exercise, smoking and alcohol consumption, also significantly affect bone density [17]. Consequently, the risk of osteoporosis cannot be solely predicted based on the BsmI polymorphism of the VDR gene.

4.3. BsmI & the efficacy of bisphosphonates

Strong bones are crucial for maintaining human health, as osteoporotic fractures can significantly increase the risk of disability or fatality. Consequently, both the prevention and treatment of osteoporosis are essential. The strategies for managing osteoporosis typically include basic preventative measures, pharmacological interventions and rehabilitation treatments. Effective pharmacotherapy can improve bone density, enhance bone quality and reduce fractures risk [17]. The primary classes of anti-osteoporosis medications include basic supplements such as alfacalcidol, bone resorption inhibitors like bisphosphonates and bone formation promoters such as parathyroid hormone [18]. Among these, bisphosphonates are the most commonly used, binding to the bone surface during active bone remodeling and inhibiting osteoclast activity, thus reducing bone resorption [18]. However, the effectiveness of these drugs can vary significantly among patients, with data showing that less than 1/4 of patients receive effective treatment, underscoring the need for personalized treatment approaches for osteoporosis patients both now and in the future. Research has shown that VDR gene polymorphism (BsmI) can influence the efficacy of treatments such as bisphosphonates and raloxifene in osteoporosis patients [19]. A study involving 68 postmenopausal women who received alendronate sodium (10 mg/d) and underwent VDR gene BsmI polymorphism typing observed significant changes in BMD after 12 months. Women carrying the b allele exhibited notably higher improvements in BMD and better treatment outcomes [20]. Additionally, in patients with Paget’s disease, those with the b allele experienced more effective treatment with clodronate compared with those with the B allele (p < 0.05) [21]. This highlights the importance of understanding genetic variations in the pathogenesis of osteoporosis across different ethnic groups, which is crucial for its diagnosis, treatment and prevention.

4.4. BsmI gene detection & its application in rational drug use in patients with osteoporosis

Before the implementation of the genetic testing project at our hospital, clinicians relied on their experience or standard drug guidelines to treat patients, which often led to unsatisfactory treatment outcomes. During this period, 14 cases of adverse drug reactions (ADRs) were recorded following the use of bisphosphonates. However, after the genetic testing project was initiated, the pharmacy department provided clinicians with medication reports for 350 cases based on the the tests results. These reports facilitated the prescription of precise, individualized medication, including bisphosphonates, tailored to each patient’s BMD and osteoporosis indicators. Consequently, there was a notable enhancement in the clinical efficacy of anti-osteoporosis treatments, with only two patients experiencing ADRs. Of the 350 patients, 18 had been hospitalized for osteoporosis prior to the commencement of genetic testing. Clinicians used the comparative data of patient’s BMD from before and after the genetic analysis to make informed treatment decisions. Following the tailored medication reports, 11 of these patients received bisphosphonate treatment. This group saw a significant improvement, in their bone mineral density scores, alleviation of symptoms related to their condition, and overall remarkable treatment effects. Conversely, there were seven patients whose bone mineral density decreased. Analysis revealed that in these cases, clinicians did not selected bisphosphonates based on genetic testing sensitivity but had opted for other treatments such as alfacalcidol or salmon calcitonin, which led to suboptimal therapeutic outcomes. Notably, these seven patients were older, ranging in age from 70 to 92 years.

The decrease in BMD was not solely due to drug selection factors, but also to variations in drug absorption and metabolism among individuals. Therefore, genetic testing plays a significance role in guiding clinical medicine toward providing individualized precision medicine. ADRs present a persistent challenge in clinical practice, as they can only be addressed after they occur, without the ability to predict them in advance. However, pharmacogenetic testing, based on pharmacogenomics, offers potential solutions to this issue [22]. For instance, genetic variations in drug metabolism genes or susceptibility genes have been linked to the prevention of ADRs. Specifically, variations in the SLCO1B1 gene can help identify the risk of myopathy in patients undergoing simvastatin therapy [23], and the presence of the HLA-B*5801 variant increases the risk of severe Stevens–Johnson syndrome and toxic epidermal necrolysis in individuals administered allopurinol [24]. Therefore, by comparison adverse reaction case data before and after genetic testing, it is evident that VDR gene polymorphism provides a certain level of guidance in clinical drug selection and in the prevention of adverse reactions. This area warrants further exploration in future research.

5. Conclusion

Despite the promising findings, several challenges remain in the clinical application of VDR gene polymorphism detection for osteoporosis treatment. One primary challenge is the variability in drug efficacy among different individuals, which highlights the need for personalized treatment strategies. Additionally, the limited studies on the distribution and impact of VDR BsmI locus polymorphism within different Chinese populations suggest a need for further research to validate these findings across diverse ethnic groups. Future research should focus on large-scale, multicenter studies to explore the relationship between VDR gene polymorphisms and osteoporosis more comprehensively. Moreover, advancements in genetic testing technologies and methodologies will be crucial to enhance the precision and applicability of genotype-based drug recommendations. The integration of pharmacogenomics into routine clinical practice promises to improve the effectiveness of osteoporosis treatments, minimize ADRs, and pave the way for personalized medicine in managing bone health. By addressing these challenges and leveraging future research opportunities, we can significantly enhance the understanding and management of osteoporosis, ultimately improving patient outcomes and quality of life.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

This study was approved by the Ethics Committee of the Lunjiao Hospital, Shunde District, approval number (2023) No.001.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Data availability statement

The simulation experiment data used to support the findings of this study are available from the corresponding author upon request.