Abstract
The aim of this study is to demonstrate the relationship between the expression of the Duffy antigen receptor for chemokine (DARC) gene in peripheral blood leucocytes and epithelial cells of the oral cavity and the level of bone mineral density (BMD) in the mandible of women during menopause. A special radiographic method for measuring mandible density was used. Studies of DARC gene expression in the epithelial cells of the oral cavity and peripheral blood leucocytes did not confirm a significant correlation between expression of DARC and the level of BMD of the mandible in patients from the study groups, both in assessing the level of gene expression (Δ C T DARC) in the epithelium and leucocytes, as well as the value of relative expression (RQ) of DARC epithelium and lymphocytes. Significant differences were found between the values of Δ C T DARC marked in leucocytes and the epithelium of the control and study groups. Regarding RQ, the tests showed no significant differences between the values marked in the epithelial cells and leucocytes. The influence of DARC gene expression on bone mineral density of the mandible was not confirmed.
Osteoporosis within the osseous structures of the stomatognathic system has been the subject of research for many years. The studies have focused mainly on the analysis of mandibular bone density, analysis of mineral content, and the structure of the jaw as a denture or implant supporting area.
The pathogenesis of osteoporosis is now understood better than it was 10–15 years ago. In addition to the mechanisms regulating bone metabolism at the cellular level through the activity of osteoblasts and osteoclasts, more is known about the molecular factors directly involved in the activation of these cells. Research on factors regulating bone metabolism is carried out in many fields such as biomechanics of bone involving cytokines, hormonal factors, and genetic tests showing which genes are involved and how they affect the structure and quality of bone.
On the basis of genetic tests, knowledge about the influence of hormones on bone metabolism has expanded to include the role of particular receptors for factors involved in bone metabolism, such as oestrogen and its two receptors, ERα and ERβ. Attempts have been made to determine the role of particular oestrogen receptors in bone metabolism. Similar studies were conducted in the context of assessing the significance of polymorphism in the vitamin D receptor and its role in the regulation of bone density. Whether the variation of these and other genes (polymorphism) results in different bone density has not been determined.
Many years of research, conducted on large groups of patients in different centres, have suggested that genetic factors influence the regulation of bone metabolism. To date, genes that have a critical impact on the pathogenesis of osteoporosis have not been found, however, many genes of minor importance in development of osteoporosis have been discovered. Within each chromosome there are genetic loci regulating bone density. In 2007, experimental studies under the leadership of Mohan revealed the presence of a gene located on chromosome 1, which plays a significant role in the pathogenesis of osteoporosis. The protein coded by the Duffy antigen receptor for chemokine (DARC) gene regulates the formation of osteoclasts. It is known that DARC protein combines with chemokines involved in the development of osteoclasts. DARC is a membrane glycoprotein and a non-specific receptor for several chemokines; it is also a receptor for the human malaria parasites ( Plasmodium vivax and Plasmodium knowlesi ). DARC protein was initially identified on the surface of red blood cells; its name comes from the name of a patient in whose blood it was first identified. DARC is present in greater quantities on reticulocytes than on erythrocytes. It has also been identified in some epithelial cells, Purkinje cells in the cerebellum, thyroid capillary endothelial cells, vascular endothelial cells of certain organs, and cells of major vessels of the lungs.
DARC protein is a transmembrane glycoprotein, a receptor for several chemokines such as CC chemokines, Ccl2, Ccl5 and interleukin-8. It has been shown that chemokines regulate the formation and activity of osteoblasts by tumour necrosis factor (TNF) family ligand (Tnfsf11) in the dependent and independent mechanisms.
The mechanism by which DARC affects osteoclasts probably occurs mainly by its affinity for chemokines involved in the metabolism of osteoclasts. In a study by Chaudhuri et al., it was confirmed that DARC participates in the process of cell migration and affects the bioavailability of several chemokines, including Ccl2 and Ccl5. These are chemotactic signals for monocytes and are responsible for the migration of osteoclast precursors and combining them in differentiated osteoclasts. According to Kim et al., activation of osteoclasts by Tnfsf1 and Csf1 leads to increased expression of Ccl2 and Ccl5, which combine with DARC.
The human Duffy blood group system is composed of four alleles, five phenotypes, and five antigens. Duffy-negative individuals, who are mainly Africans and African-Americans, do not have DARC protein on the surface of erythrocytes and are resistant to P. vivax and P. knowlesi . African-Americans exhibit significantly higher bone mineral density (BMD) compared to Caucasians.
Currently available sources show that the DARC gene coding DARC protein is involved in the regulation of BMD by controlling the genesis of osteoclasts. In studies on experimental models, it was confirmed that the exclusion of the DARC gene resulted in decreased femoral bone resorption in mice. This confirms that DARC is the regulator of genesis of osteoclasts and its deficiency results in a reduction of bone resorption, thereby increasing BMD. There are no reports about the expression of the DARC gene in the oral mucosa, but the role of the DARC gene in bone regulation has been confirmed in experimental studies, encouraging investigation into mandible BMD and DARC gene expression in the epithelium of the human oral mucosa.
This study was designed to designate DARC gene expression and to determine its possible impact on mandibular BMD in menopausal women. The mandible was chosen as a valuable bone for DXA examination. The DARC gene, previously known as CD234, codes a membrane protein which is a non-specific receptor for several chemokines such as IL-8, GRO, RANTES, MCP-1 and TARC. The DARC gene has been linked to the group of genes directly related to osteoporosis.
The aim of this study is to demonstrate the relationship between the expression of the DARC gene, coding the protein in peripheral blood leucocytes and the epithelial cells of the oral cavity, and the level of BMD in the mandible of women during menopause compared with a control group of women aged 23–25 years.
Demonstrating possible differences in gene expression between these groups and their correlation with BMD would confirm the significance of DARC gene expression in the diagnosis of osteoporosis in the stomatognathic system (the mandible), and confirm oral epithelial cells as valuable material for genetic tests.
Materials and methods
The research was conducted on two groups of patients. The study group included 30 menopausal women (at least 1 year after last menstruation, aged 55–65 years), not taking hormone replacement therapy, and generally healthy. The control group consisted of 30 women aged 23–25 years, who were generally healthy, non-smoking, and represented peak bone mass age in women.
Analysis of gene expression of DARC, according to the procedure specified by the manufacturer, was conducted. The study of mandibular BMD was performed using a DXA device (GE Healthcare Lunar Prodigy Advance™). The mandible in the premolar region was chosen as a referral region for examination to obtain unilateral images of the mandible corpus area. There is no standard equipment for carrying out this type of research in the mandible. Patients were put into a position corresponding to the position of patients in the conventional lateral oblique projection of the mandible to avoid overlapping the structures of the right and left side of the mandible ( Fig. 1 ). The results were subjected to statistical analysis to assess the effect of gene expression on the BMD of the mandible.
The clinical dental examination was conducted in the study group (average age 55.4 years). The average age in the control group was 24.2 years. All patients gave their consent to examination and the testing procedures received approval from the Local Ethics Committee in Lublin. No of decision KE-0254/140/99, KE-0254/246/2001 and KE-0254/47/2010.
Patients from the study group were qualified for examination after the interview and underwent clinical examination. Blood samples were taken from women in a fasting state, and were submitted for genetic research to the Laboratory of Clinical Genetics, Medical University of Lublin, where blood lymphocyte isolation was carried out to study gene expression: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) – control gene, DARC. Using a sterile laryngology spatula, epithelium was collected from the buccal mucosa, resuspended in 0.9% NaCl and isolated by centrifugation to examine the expression of selected genes in the epithelial cells of the mouth.
After collecting the material for genetic testing, patients were qualified for radiological examination (densitometry of the jaw). Mandibular bone density tests were performed with a densitometer (GE Healthcare Lunar Prodigy Advance™) using X-ray absorption in materials with different densities. The device emitted radiation in two energy canals. Images, performed on the right and left side of the mandible, were scanned using dual-energy X-ray absorptiometry (DXA). An area of interest (corpus of the mandible in the area of the mental foramen) was selected, and the computer calculated the average bone density of the area. After obtaining a result, the arithmetic mean was calculated for each patient with data obtained from scans of the right and left side of the mandible. In order to obtain an image of the corpus of the mandible in the examined area, the appropriate positioning of patients during the densitometric examination was used. There is no standard unit prepared to carry out this type of research on the mandible. In order to avoid overlapping of the structures of the right and left side of the mandible, patients were put into a position corresponding to the position of patients in the conventional lateral oblique projection of the mandible.
Genetic laboratory tests
Blood was collected from the ulnar vein and epithelium was gathered from the vestibulum of the oral cavity to test gene expression. For each patient, from the study and control groups, two separate indications of gene expression were performed. Further analysis was conducted with the use of the arithmetic mean obtained for each patient.
Isolation of total cellular RNA was carried out with the modified method of Chomczynski and Sacchii using Sigma reagent TriReagent, according to the manufacturer’s procedures. The following sets of probes were used: type TaqMan marked with FAM-NFQ and primers for genes; and DARC and GAPDH as an endogenous control (AppliedBiosystems). The reaction consisted of the following thermal cycles: initial denaturation 95 °C for 10 min, 40 cycles; 95 °C 15 s; 60 °C 60 s. The number of copies of gene molecules of nucleic acid was monitored in each cycle of amplification. A number of polymerase chain reaction (PCR) cycles, after which the level of fluorescence exceeded the defined threshold (TCT), were used to calculate the amount of tested molecules present in the mixture at the beginning of the reaction (analysis using the software RQStudy; AppliedBiosystems). For each trial, the value of C T for the GAPDH gene (endogenous control) was used to the level of normalization of gene expression.
The levels of gene expression (Δ C T ) were determined by the formula:
Δ C T of gene = C T of gene − C T GAPDH
Relative expression (RQ) of the studied genes was determined by the formula:
RQ = 2 − ( Δ C T of gene − Δ C T of calibrator )
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