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GIPR rs10423928 and bone mineral density in postmenopausal women in Shanghai

We demonstrated previously that there is a correlation between glucagon-like peptide-1 (GLP-1) single-nucleotide polymorphism (SNP) and bone mineral density in postmenopausal women. Both GLP-1 and glucose-dependent insulinotropic peptide are incretins. The glucose-dependent insulinotropic peptide receptor (GIPR) SNP rs10423928 has been extensively studied. However, it is not clear whether GIPR gene mutations affect bone metabolism. The aim of this study was to investigate the association between rs10423928 and bone mineral density in postmenopausal women in Shanghai. rs10423928 was detected in 884 postmenopausal women in Shanghai, and the correlation between the GIPR SNP and bone mineral density was assessed. The dominant T/T genotype of rs10423928 was found to be related to the bone mineral density of the femoral neck (P = 0.035). Overall, our findings indicate that the dominant T/T genotype of rs10423928 in postmenopausal women is significantly associated with a higher bone mineral density and that the T/T genotype exerts a bone-protective effect.

Glucose-dependent insulinotropic peptide (GIP) plays a biological role by binding to the corresponding GIP receptor (GIPR) on the cell surface. GIPR is located on chromosome 19 q13.3 and comprises 14 exons, having the size of approximately 14 kb (12). GIPR, which is widely expressed in various tissues and organs, is also expressed in extra-pancreatic cells, such as osteoblasts and the small intestine (13,14). Mutations in GIPR may lead to abnormal GIPR expression and function, which may be related to the risk of osteoporosis. Furthermore, phenotypic differences in the human body and susceptibility to drugs or diseases may be related to single-nucleotide polymorphisms (SNPs). There are 13 SNPs in GIPR. Among them, rs10423928 has been studied extensively. It involves the replacement of thymine with an adenine base (T/A) (15). Research on rs10423928 mainly focuses on type 2 diabetes and impaired insulin secretion (16,17) Both GLP-1 and GIP are incretins. Previously, we demonstrated a correlation between GLP-1 receptor gene (GLP-1R) polymorphisms and BMD in postmenopausal women in Shanghai, China (18). Therefore, we aimed at elucidating whether there is a correlation between GIP/GIPR and BMD and whether GIPR mutations affect the effects of GIP/GIPR.

Subjects
The study was approved by the Ethics Committee of the Sixth People's Hospital, Shanghai Jiaotong University (2014-KY-001(K)). Han women who are being treated for osteoporosis at Shanghai Sixth People's Hospital were enrolled in this study. All subjects signed informed consent forms. BMD and other clinical data were analyzed.
The inclusion criteria were as follows: (i) natural menopause for more than 1 year and (ii) no anti-osteoporotic treatment (except for calcium and vitamin D supplementation). The exclusion criteria were any disease that affects bone metabolism and use of medication, except for GLP-1 analogs or DPP4 inhibitors.
We included 907 postmenopausal women in this study; however, in order to minimize selection bias, some subjects were excluded, some samples were contaminated, were of poor quality, or were not successfully typed after one failure. Finally, 884 samples from postmenopausal women (mean age: 67.2 ± 10.0 years) were subjected to SNP detection. The average menopausal age of the 884 postmenopausal women was 47.41 years, and 90% of them had osteopenia/osteoporosis and 307 women had a history of fractures. These 307 women were ruled out of diseases and external factors that affect bone metabolism, accounting for approximately 34.73% of the participants.
Amplification was achieved by performing multiplex PCR. Each measurable allele locus-ligation product was obtained after two ligation reactions. The raw data files were analyzed using the GeneMapper 4.1 software (Applied Biosystems). The iMLDR® multiple SNP typing system (Shanghai Tianhao Biotechnology Co., Ltd., Shanghai, China) (19) was used to classify the GIPR rs10423928 SNP site in 884 samples from postmenopausal women.

BMD
The BMD of the lumbar spine 1-4 (L1-4), left femoral neck, and total hip was measured for postmenopausal female subjects, expressed in g/cm 2 , using dualenergy x-ray absorptiometry (GE Lunar Prodigy Bone Densitometer, Little Chalfont, UK). Following strict quality-control requirements, the instrument was tested once daily before use with a standard phantom to evaluate the stability of the system. The coefficient of variation of the lumbar spine, left femoral neck, and total hip BMD measurements was 1.39, 2.22, and 0.70%, respectively.

Statistical analysis
Statistical analysis was performed using the SPSS software (version 24.0; SPSS, Inc.). Continuous variables with a normal distribution are expressed as mean ± s.d. (x ± s). Continuous variables between groups were compared using the t-test. Chi-squared tests were used to compare categorical variables. The Haploview software (version 4.2) was used to calculate the D' value and LD coefficient (r 2 ) between SNPs. After adjusting for age, linear regression was used to assess the relationship between GIPR SNP and BMD in postmenopausal women. P < 0.05 was considered to reflect a statistically significant difference.

Results
To determine the effect of aging on osteoporosis in elderly women, the subjects were divided into different age groups according to a recognized age classification system (<60 and ≥60 years) (20). Table 1 shows the baseline characteristics of these subjects. No significant difference was detected in the serum calcium and phosphorus levels between the groups. Alkaline phosphatase (AKP) was significantly higher in the group aged ≥60 years than in the group aged <60 years (P = 0.004). The BMD in the group aged ≥60 years was lower than that in the group aged <60 years (P < 0.001).

Allele frequencies
The GIPR rs10423928 locus was analyzed. The genotype distribution met the Hardy-Weinberg equilibrium. The MAF of rs10423928 in the CHBS (representing the national database) is 20%, and in the present study, it was 20.8% (Table 2). There was no linkage disequilibrium relationship between rs10423928 and GLP-1R SNPs (0.908 < D' < 1) (Fig. 1). Table 3 shows the clinical data corresponding to rs10423928 in postmenopausal women. The rs10423928 locus has three genotypes: T/T, A/T, and A/A. Among them, the T/T genotype was detected in 556 patients (62.9%), the A/T genotype was detected in 289 patients (32.7%), and the A/A genotype was detected in 39 patients (4.4%).
Based on the linear regression analysis, the rs10423928 locus was unrelated to age, height, weight, BMI, Ca, P, PTH, AKP, ALB, and Scr (P > 0.05; Table 4). A close correlation was noted between the rs10423928 locus and BMD. The T/T genotype positively correlated with the femoral neck BMD and Ward's triangle area BMD in postmenopausal women (P < 0.05; Table 5).

Discussion
Genome-wide association analysis has confirmed that BMD is associated with multiple genetic-susceptibility regions (21,22). Correlations between vitamin D receptor gene polymorphisms and osteoporosis were discovered in 1994 (23). Subsequently, >100 gene polymorphisms related to bone metabolism regulation have been identified.
Currently, there are no relevant data from clinical research showing that GIPR polymorphism is related to bone metabolism. In our previous research, we found that GLP-1R SNP is related to bone metabolism. Both GIP and GLP-1 are incretins, and both of them are related to glucose and lipid metabolism. The purpose of our research was to determine whether there is a correlation between GIPR SNP and bone metabolism.
Some clinical and animal studies have shown that GIPR has a relationship with bone metabolism. Patients with type 2 diabetes express low levels of GIPR or have defective GIPR (24,25). In GIPR knockout mice, the cortical bone becomes thinner, the number of endosteal osteoclasts increases, and BMD decreases, indicating that there is a decrease in bone strength and bone quality (11). GIPR knockout mice exhibited an increased plasma calcium concentration after feeding, indicating that GIP may play a role in calcium homeostasis (26). GIP can guide the absorption of skeletal nutrients through the gutbone axis and regulate bone turnover. There is a close relationship between bone turnover and intestinal hormones (27,28,29).
GIP can affect bone metabolism directly and indirectly (30,31,32,33,34), and it plays a biological role by binding to the corresponding GIPR on the cell surface. GIPR SNP rs10423928 has been studied frequently. It consists of the major allele T and the minor allele A. Individuals with the T/T genotype who consume a high-carbohydrate/low-fat diet have a lower risk of type 2 diabetes (16). The minor allele A is associated with an impaired insulin secretion stimulated by glucose and GIP and a decreased BMI, lean body mass, and waist circumference (17). In the present study, we found that the BMD in the group aged ≥60 years was lower than that in the group aged <60 years (P < 0.001). This indicates that with age, the bone mass of women decreases (35,36). We neither find that the genotypes of rs10423928 are associated with BMI and body weight nor did we find an association of each genotype with Ca, P, PTH, and AKP.
Studies have found that there is a strong linkage disequilibrium between the SNPs rs10423928 and rs1800437 (Glu 354Gln located in exon 10, E354Q) of the GIPR locus (r 2 = 0.99). rs1800437 can reduce the expression of GIPR in carriers (37,38). In the Danish Osteoporosis Prevention Study (39), rs1800437 was associated with BMD and fracture risk. Compared with those with the major allele G, the femoral neck BMD and total hip bone BMD of women with the minor allele C were significantly reduced. Women who are homozygous for the variant C allele have an increased risk of non-vertebral fractures. Our study revealed that the dominant T/T genotype of rs10423928 in postmenopausal women was significantly associated with a higher BMD of the femoral neck. This indicated that the T/T genotype of GIP has a protective effect against osteoporosis.
Our study was not a prospective study but a crosssectional cohort study. We found that the dominant T/T genotype of rs10423928 was significantly associated with a higher BMD, unlike the other genotypes in postmenopausal women. This does not indicate that this genotype can prevent or warn of osteoporosis. The T/T genotype seems to have a bone-protective effect, but the mechanism needs to be further explored.
Taken together, our study showed that the dominant T/T genotype of rs10423928 in postmenopausal women is significantly associated with a higher BMD and that the T/T genotype seems to have a bone-protective effect.

Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding
This work was supported by the Pudong New Area Health and Family Planning Commission Fund Project (grant number PWZzk2017-12).

Ethical statement
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Ethics Committee of the Sixth People's Hospital, Shanghai Jiaotong University (2014-KY-001(K)).  GIP receptor gene polymorphism and BMD e210583 11:2 X Sun, W He, Dongyue Pang, Jingjing Hu. All authors contributed to writing the manuscript and approving the final manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.