Osteoporosis (OP) is a skeletal disease characterized by decreased bone mass and impaired microarchitecture leading to decreased mechanical strength and increased fracture risk (1). It has become a significant global public health concern due to the increasing number of fractures and the negative impact on the quality of life of affected individuals (2). Recent reports indicate that about 75 million people in Europe, the United States, and Japan are affected by OP, of which about 8.9 million have suffered fragility fractures. In Mexico, according to data from the 2020 population and housing census, the current population is around 126 million inhabitants, from which 17.4% correspond to individuals the subpopulation aged 50 years or older. It is estimated that about 10 million people in Mexico are living with OP (3, 4).

This disease is characterized by being multifactorial and complex where the predisposition, pathogenesis, or response to treatments are modulated by the interaction between genetic and environmental factors (5). The heritability of OP has been reported as high 50–85% (6, 7). The most effective approach for detecting the Single Nucleotide Variants (SNVs) associated to a multifactorial condition is through genome-wide association studies (GWAS). Therefore, several studies have aimed to link SNVs across the genome with the occurrence of OP, with the goal of identifying individuals at higher risk (8).

Microtubule affinity-regulating kinase 3 (MARK3) gene encodes a serine/threonine kinase, which is activated by the hepatic tumor suppressor kinase B1 (LKB1) and antagonizes oncogenic pathways, including the cell cycle pathway through phosphorylation of CDC25C (9). Regarding bone metabolism, a GWAS identified a locus containing multiple genes, including MARK3, TRMT61, and CKB. The main SNV associated was rs11623869, which lies in the second intron of MARK3. This locus was associated with femoral neck (FN) and lumbar spine (LS) BMD, and consistently replicated in Chinese and European populations (10, 11). A recent study implicates this kinase as an important signaling molecule in osteoblasts influencing bone mass (12).

Another study carried out in Taiwanese population identified the SNV rs6086746 upstream the Phosphoinositide Phospholipase C-Beta-4 (PLCB4) gene associated with low BMD in postmenopausal women. PLCB4 encodes a phospholipase C which participates in the phosphoinositide cycle signaling pathway, transmitting information from the extracellular environment into the cell, influencing several cellular processes. It has been reported that rs6086746 may affect the binding of the transcription factor RUNX2 to the promoter region of PLCB4, this might be part of the mechanisms contributing to the development of OP (13). RUNX2 is a key transcription regulatory factor in osteoblast differentiation, it plays an important role in regulating osteoblast maturation and balance (14). Several studies reported that polymorphisms on the promoter of RUNX2 are associated with BMD, in Korean and European populations (15–17).

The Gemin protein associated with the nuclear organelle 2 (GEMIN2) is part of a complex composed by the survival motor neuron protein (SMN) and seven additional Gemin proteins (GEMIN2–8). This complex is mainly involved in the assembly of the small nuclear ribonucleoprotein (snRNP) machinery, which regulates mRNA splicing in the cytoplasm (18). In particular, the SMN complex functions as a molecular chaperone whose phosphorylation regulates the biogenesis and function of snRNPs involved in mRNA splicing (19). Additionally, the SNV rs2277458 of the GEMIN2 gene has been reported to be associated with the variation in plasma concentrations of 25-hydroxyvitamin D in the Danish population. In this population, low plasma concentration of 25-hydroxyvitamin D has been associated with a higher risk of osteoporotic fractures (20). Based on current evidence, this study aimed to investigate the effect of three recently identified SNVs (MARK3-rs11623869, PLCB4-rs6086746, and GEMIN2-rs2277458) on BMD, in a cohort of Mexican women. Additionally, we explored the effect of the interaction between these genetic variants with serum 25(OH)D levels and BMD.

Our study confirmed previous evidence indicating associations between the genetic variants MARK3 (rs11623869), PLCB4 (rs6086746), and GEMIN2 (rs2277458) with osteoporosis and serum 25OHD levels in a European-descent population and a southern Chinese cohort also associated with an admixed population such as the Mexican-Mestizo. These findings suggest that the genetic factors influencing osteoporosis and vitamin D levels may transcend ethnic boundaries, emphasizing their relevance across diverse population groups.

In recent years, it has been reported that circulating factors such as calcium, phosphate, 25(OH)D, PTH and ALP, have been related to the variation of DMO. They can act directly or indirectly on skeletal cells, regulating bone remodeling and metabolism. Vitamin D is essential for efficient calcium absorption. An adequate amount of vitamin D is required to maintain bone strength and prevent fragility fractures (22) Vitamin D deficiency (25OHD <20 ng/mL) is common in Mexican older adults and linked with factors such as sex, age, genetics, diet and obesity (23, 24). In addition, the lower intake of vitamin D and calcium reported in the Mexican population (25) can cause inefficient absorption of calcium, which in turn can stimulate the release of calcium from the bones to maintain a normal concentration of calcium in the blood and consequently cause bone loss. Decreasing serum calcium levels can stimulate PTH secretion, and PTH in turn can improve serum calcium concentration by releasing calcium from bone by increasing bone resorption. However, several studies have reported the negative impact of elevated serum PTH levels on BMD (26). Calcium and phosphate are co-dependent for bone development, a sufficient amount of phosphate in the blood has a positive effect on calcium use and adequate bone growth (27), while ALP plays an important role in the formation and mineralization of osteoid and is used as a biomarker to evaluate bone turnover. The high rate of bone turnover in elderly people can lead to rapid bone loss and reduced bone mass. Interactions between genetic, dietary, hormonal, metabolic, and lifestyle factors have been suggested to play an important role in susceptibility to low BMD. In this sense, the risk caused by genetic variants may vary between individual’s due to differential modifications of the different concentrations of circulating factors involved in bone remodeling and metabolism. While a study by Xiao SM, et al. in 2013 reported an association between the SNV rs11623869 of the MARK3 gene and BMD, particularly strengthened in the presence of high serum levels of ALP (22), no association studies have been conducted for the SNVs rs6086746 in PLCB4 and rs2277458 in GEMIN2 with serum variables such as vitamin D, calcium or ALP, and their potential effects on BMD variation.

It has been reported that the MARK3 gene is involved in different biological processes such as cell cycle, ciliated cell differentiation, and osteoclast differentiation (28). So far, the role of MARK3 has been studied in several pathological conditions (9, 29–31). In osteoporosis, the variant rs11623869 in MARK3 has been associated with bone mineral density and low-trauma fractures (32, 33). Our results showed that the rs11623869-T variant is associated with low BMD at the total hip. These data are consistent with previous reports where the T allele was associated with a decreased BMD at femoral neck accompanied by an increased expression of MARK3 (32). The authors showed that Mark3-deficient osteoblasts exhibited an increase of bone mass, through reduced Jag1/Hes1 expression and decreased downstream JNK signaling stimulating osteoblast activity. On the other hand, in Chinese population, it was observed that the effect of the SNV rs11623869 on BMD was greater in the presence of high serum levels of ALP, a biomarker for osteoblast activity, and considered a predictor of BMD in postmenopausal females (33). Together these data suggest that genetic variation of MARK3 influences bone mineral density. However, current data also suggest that circulating factors in serum may also affect bone turnover and metabolism modifying the association of MARK3 with BMD. Further research is necessary to clarify the role of these circulatory factors in Mexican postmenopausal women.

Another gene of interest is PLCB4, its product catalyzes the formation of inositol 1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate, using calcium as a cofactor. PLCB4 plays an essential role in signal transduction (34). Genetic variants in PLCB4 have been identified as cause of the Auriculocondylar syndrome 2 (ARCND2), a disease characterized by craniofacial malformations (13), PLCB4 acts as a direct signaling effector of the endothelin receptor type A (EDNRA)-Gq/11 pathway. Kanai et al, 2022 demonstrated that variants in PLCB4 gene interfere with the EDNRA signaling pathway, leading to the development of ARCND2 (35). However, the role of genetic variants in PLCB4 on BMD has been explored only to a limited extent. Only one study in the Chinese population reported that carriers of the A alleles of the rs6086746 variant in PLCB4, showed a decrease in BMD and an increased risk of developing osteoporosis. Furthermore, they found that the rs6086746 variant was significantly associated with osteoporosis, by participating on the binding of RUNX2, a master transcription factor involved in the osteoblast maturation. Through luciferase assays, they showed increased to that PLCB4 activity in individuals carrying the rs6086746-A allele, proposing a functional explanation for the observed association (13). In contrast, our results shown that variant rs6086746 was associated with higher BMD and protective effect against low-BMD in women aged 45 years or older. These results are consistent with an independent group of OP patients from the National Institute of Rehabilitation (INR) in Mexico City (36). Although the frequencies were similar in both cohorts, we did not observe statistical differences (OR = 0.74, 95%CI 0.47-1.16), perhaps due to the small sample size (n=384).

Recently, three genotypes associated to plasma 25-hydroxyvitamin D levels were identified through genome-wide association studies; among which is the variant rs2277458 in GEMIN2 (37). GEMIN2 encodes a protein of the SMN complex, this complex includes several Gemin proteins and the SMN protein. The SMN complex is located at a subnuclear compartment called gems (Gemin of coiled bodies) and is necessary for the assembly of spliceosomal snRNPs and for pre-mRNA splicing. Although there is no evidence implicating GEMIN2 in vitamin-D-related physiological pathways.

More recently, in a study in the Danish population, the rs2277458-A allele of GEMIN2 was associated with lower serum 25-hydroxyvitamin D concentrations in a dose-dependent manner (20). These data are consistent with our results where the rs2277458-G allele of GEMIN2 was associated with lower serum 25-hydroxyvitamin D levels, in women aged 45 years or older. Low vitamin D concentrations are believed to affect bone metabolism by decreasing dietary calcium and phosphorus absorption and increasing the production of parathyroid hormone (38). In addition, vitamin D activates osteoblasts and osteoclasts to dissolve the mineralized collagen in bones, causing osteopenia and osteoporosis, thus increasing the risk of fractures. Together these data suggest that genetic variants in GEMIN2 influences serum 25-hydroxyvitamin D concentrations, in different populations (39).

Vitamin D has been shown to play a fundamental role in calcium and phosphate homeostasis, furthermore, serum levels of vitamin D are positively correlated with BMD values (40). In contrast, vitamin D deficiency causes a reduction of BMD, increasing the risk of bone fractures in the elderly (41). In our study, the genetic risk score constructed using the three variants (equivalent to 3 risk alleles) was significantly associated with higher BMD at the hip and femoral neck, as well as elevated serum 25OHD levels. These associations were particularly pronounced among postmenopausal women aged 45 years and older. This suggests that the cumulative genetic risk, as represented by the GRS, may contribute to improved bone health and vitamin D status in this population. These results are consistent with those reported by Mithal, et al., 2009, who analyzed a population from Latin America including Mexico that was composed of postmenopausal women and in which they observed that vitamin D levels are below of the average values, due to geographical and population characteristics (23, 42).

A possible explanation for these findings is that vitamin D consumption could improve BMD levels in women over 45 years of age with higher genetic risk, given that the associated genetic variants are linked to vitamin D levels in the body. Additionally, the SNVs included in the genetic risk score may influence vitamin D metabolism, thereby strengthening the response. Therefore, these data may generate a new focus on the possible role of these SNVs on vitamin D metabolism. Nevertheless, these data should be taken with caution, as a larger and more diverse population is needed for confirming or discarding these findings.

The literature regarding the interaction between genes related to vitamin D levels and BMD, in Mexican Mestizo population is scarce (43). This study provides evidence suggesting that this phenomenon could be participating in the high prevalence of vitamin D deficiency in the Mexican population. Furthermore, interactions between genes are proposed to serve as a key factor involved in the variance of BMD. We have identified an effect of the interaction between SNV rs11623869 in the MARK3 gene and the SNV rs22277458 in GEMIN2 on hip and femoral neck BMD. The influence of these interactions on BMD is complex, concentrations of circulating factors related to calcium and phosphate metabolism might alter the effect of genetic risk factors and the subsequent loss of BMD. Studies considering the interaction of genes involved in BMD loss in conjunction with known factors involved in calcium and phosphorus metabolism will contribute to unravel this complex relationship. Furthermore, interactions between genetic and lifestyle factors have been suggested to play an important role on susceptibility to having low BMD and serum 25OHD levels. The risk caused by genetic variants may vary between populations due to exposition to different environmental factor and lifestyle habits. In this study, we found that the association of the rs22277458 in GEMIN2 with serum 25OHD levels was strengthened in the presence of the rs6086746 PCLB4. However, the role of GEMIN2 and PCLB4 on vitamin D metabolism remains unknown. Also, we cannot exclude that variants in these genes may only indirectly affect 25 hydroxyvitamin D through pathways affecting both, exposure and outcome separately. Further, we cannot exclude the possibility that other mechanisms involving these genes may exist. Finally, the association of genetic variants in GEMIN2 and PCLB4 genes with serum 25OHD could represent a chance finding and, therefore, needs additional confirmation in an independent cohort.

This study has some strengths: first this analysis was conducted in a relatively large sample (n=1300) compared to other observational studies. Second, this is the first study focused on understanding the effect of genetic variants involved in vitamin D metabolism and bone mineral density in postmenopausal women. On the other hand, this study has some limitations. First, this work did not analyze GRS with serum calcium levels and bone fracture as reported in other studies. This could strengthen our hypothesis about the effect of SNVs of genes involved in vitamin D metabolism. Furthermore, since our study is cross-sectional, we cannot establish causality between SNVs, vitamin D levels, and BMD. In this work, it will be observed that vitamin D consumption is positively associated with BMD and is essential for efficient calcium absorption. However, a limitation of this study is that the relationship between serum calcium, ALP, and phosphate with BMD was not analyzed, these variables that have previously been reported for their association with bone growth and adequate osteoid mineralization. Furthermore, we acknowledge that the interaction observed between MARK3 and vitamin D levels may require replications in other independent populations, and functional studies are necessary to investigate whether the effect of MARK3 on BMD is regulated by vitamin D levels. Another limitation is that our study represents the first report showing the association between the SNVs rs6086746 in PLCB4, and rs2277458 in GEMIN2 with vitamin D levels and BMD in a Mexican population, as these genes and their respective variants had not been previously related to bone metabolism, and there are no reports on their functional role. In addition to these limitations, show the need for future longitudinal research that can address these issues. Second, we did not adjust for multiple comparisons due to the effect size found, although in recent years it has been reported that adjustment by multiple testing controls overall type I error but significantly increases type II error (44).

Our study has provided independent replications of the associations reported for the MARK3 (rs11623869), PLCB4 (rs6086746), and GEMIN2 (rs2277458) genetic variants with BMD and serum 25 hydroxy-vitamin D in a Mexican mestizo population. These results suggest that that genetic variants in these three genes may confer susceptibility for changes in BMD and serum 25 hydroxy vitamin D levels in Mexican-Mestizo, Chinese, and European-descent populations. Furthermore, we have found that SNVs rs22277458 in GEMIN2, rs11623869 in MARK3 and rs6086746 in PLCB4, highlighting the complexity of genetic and environmental factors in determining bone health. These findings have important clinical and public health implications as they could help improve the prevention, diagnosis, and treatment of bone diseases such as osteoporosis in the Mexican population. However, further longitudinal research is required to fully understand the underlying mechanisms and clinical implications of these genetic associations.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Ethics Committee from Mexican Social Security Institute (No. 12CEI 09 006 14), and the National Institute of Genomic Medicine (399–17/2016/I). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

DA-B: Writing – original draft, Writing – review & editing, Investigation, Visualization. RJ-O: Investigation, Visualization, Writing – original draft, Writing – review & editing. AB-C: Writing – review & editing, Methodology. AA-G: Writing – review & editing, Funding acquisition. VL-S: Writing – review & editing, Methodology. LC-A: Methodology, Writing – review & editing. JS: Funding acquisition, Writing – review & editing. AH-B: Funding acquisition, Writing – review & editing. BR-P: Data curation, Formal analysis, Writing – review & editing. RV-C: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.