Background
Bisphosphonates (BPs) are antiresorptive agents that are commonly used to treat osteoporosis, multiple myeloma, and metastatic solid tumors [
1]. BPs become internalized into osteoclasts via endocytosis and subsequently inhibit their activity [
2]. Despite BPs being well known to be effective in preventing osteoporotic fractures and preventing cancer-related side effects in bone, there has been a sharp decline in BP prescriptions in recent years, from 21.3 million in 2002 to 14.7 million in 2012 in the United States, with increasing reports of diverse rare but serious side effects associated with the use of BPs [
3]. Osteonecrosis of the jaw (ONJ) is one of the most-serious and well-known side effects of BPs [
4]. ONJ is characterized by jawbone necrosis, which exposes necrotic bones through holes in mucous membranes or the facial skin, traditionally ranging from a few millimeters to a few centimeters [
2,
5]. The reported incidence rate of ONJ when using BPs to treat cancer has ranged from 0.7 to 10.3% [
6], while its incidence in osteoporosis has not been established [
5,
7]. Because other drugs are also known to be risk factors for ONJ, such as other types of antiresorptive drug (denosumab) and antiangiogenics, the American Association of Oral and Maxillofacial Surgeons committee recommended to change the nomenclature of ONJ caused by drugs from BRONJ (bisphosphonate-related osteonecrosis of the jaw) to medication-related osteonecrosis of the jaw (MRONJ) in 2014 [
8].
Apart from the well-known direct causes of MRONJ such as dental surgery or gingival infection, the main mechanism underlying the occurrence of MRONJ has not been clearly elucidated [
9]. Since ONJ occurs only in specific individuals, multiple studies have been carried out to confirm the genetic background of MRONJ [
9‐
11]. Despite the dearth of genomic studies and their results not being well replicated, these studies have added a deep pathological understanding of and insight into the development of MRONJ, such as the patient’s innate immunity, angiogenesis inhibition, osteoclast suppression, and systemic/local inflammations being strong predisposing factors [
8]. Some of the candidate genes identified by these studies are
TGFb1,
MMP2,
PPARG,
CYP2C8,
VEGF,
COL1A1,
RANK,
OPG,
OPN, and
RBMS3 [
12‐
16]. However, most of these studies were either candidate-gene studies or genome-wide association studies (GWASs) [
17]. Previous whole exome sequencing (WES) studies have found that multiple biological pathway contribute to the occurrence of MRONJ, but no specific contributing genes have been identified [
9]. Recently, a study included total 44 multiple myeloma and 17 solid tumor BRONJ patients of European ancestry using WES was identified protective SNPs with significant linkage disequilibrium with
SIRT1 and
HERC4 genes [
15,
18].
In this study we applied case–control methods that are commonly used in genomics research on complex diseases to identify genes exhibiting large variations between BRONJ patients and healthy control subjects. We divided BRONJ patients into two groups depending on whether BPs had been prescribed for cancer and osteoporosis, based on the assumption that the genetic vulnerabilities contributing to the occurrence of BRONJ differ between the long-term accumulation of BPs in osteoporosis and the high-dose toxicity of BPs in cancer.
Discussion
This study evaluated the genes associated with the predisposition to develop BRONJ by comparing patients according to the reason for them being prescribed BPs (cancer or osteoporosis) using WES. The genes identified in our study—
LTBP1,
PZP,
ARID2, and
HEBP1 in osteoporosis BRON patients—clearly support the previous evidence that angiogenesis, osteoclast activity, bone remodeling, and immune responses are critical underlying mechanisms. In osteoporosis BRONJ group, we identified the
VEGFA gene which is known to play a significant role in angiogenesis was also found in previous studies to be associated with the risk of ONJ [
11]. We also identified a novel gene associated with the risk of BRONJ that is involved in angiogenesis in patients of cancer BRONJ,
PLVAP, which is the
VEGFA downstream signaling target involved in the structure of the diaphragm and functions in vascular fenestrations [
32]. Other genes identified in cancer group also have ONJ related functions: the
PZP and
LTBP1 genes are involved in TGF-β signaling (which plays an important role in bone remodeling and tissue repair), the
HEBP1 gene is involved in heme pathophysiology, and the
ARID2 gene is involved in osteoblast differentiation. These findings suggest that exposure to high-dose BPs in patients with cancer with dysfunctional genes with various underlying pathophysiologies of ONJ increase the risk of BRONJ occurrence. On the other hand, in the osteoporosis BRONJ with a relatively long-term exposure to BPs (42.7 ± 46.3 months), there were no more candidate genes to explain the pathophysiology besides
VEGFA.
Previous studies investigated to identify the contributing genetic profile of BRONJ development include two GWAS studies [
10,
33], eleven candidate gene studies [
11‐
15,
17,
34‐
38], and two WES studies [
9,
18]. Most of these studies were case–control studies involving less than one hundred single-race patients for BRONJ in cancer patients. The candidate genes and SNPs identified through these studies varied and rarely replicated in another. The pathogenesis of BRONJ is not clearly defined, however, some hypothesis has been suggested [
39,
40]. First, BPs strongly inhibits the activity of osteoclasts and induced apoptosis of osteoclasts. This reduces both bone absorption and formation. Second, BPs inhibit angiogenesis reducing blood vessel distribution in the bone along with inhibiting endothelial growth factor, which interferes with bone remodeling and wound healing in the jaw bone. Lastly, owing to the strong affinity of BPs to hydroxyapatite and long half-life leads to extreme suppression of bone turnover as well as wound healing. As the results of our study suggested as well, damaged genes involved in different but still diverse underlying mechanisms might contribute to the development of BRONJ with diverse mechanisms especially in patients with distinct underlying diseases with very different dosage and potency of BPs.
We used three analysis pipelines to identify candidate genes in order to minimize the false negative caused by various effects of causative genes and genetic variations contributing to BRONJ. The statistical technique using the traditional genetic model showed that the stop-gain mutation (rs117889746) in exon 15 of the
PZP gene was significantly associated with the occurrence of BRONJ in 2 of the 13 cancer patients (1 homozygous and 1 heterozygous). These two patients developed ONJ after receiving Zoledronate injections for 10 months and 24 months after dental procedures such as extraction and implant removal. The
PZP protein as a pan protease inhibitor is involved in the main mechanisms underlying the development of BRONJ: bone formation and inflammation.
PZP protein is similar to α2-macroglobulin and has a high affinity with TGF-β1 and TGF-β2. Binding by
PZP prevents TGFs from binding to cell-surface receptors, which in turn can eliminate TGF-β according to the morphological changes in
PZP, and also act as a carrier [
41]. TGF-β promotes tissue repair by enhancing the transcription of type I collagen, which is the main component of the extracellular matrix (ECM). Previous studies have shown that the expression of TGF-β is significantly reduced in specimens obtained from patients with nontraumatic osteonecrosis of the femoral head [
42]. This is consistent with previous immunohistochemistry studies of the TGF-β1 signaling molecule in BRONJ patients showing significantly reduced TGF-β1 and Smad-2/3 in BRONJ patients compared to osteoradionecrosis patients [
43]. Previous studies have shown that TGF-β promotes bone resorption of the mouse calvariae bone resorption at low doses and does not promote the resorption of the long bones at high doses [
44]. Therefore, the results of our study suggest that the TGF-β signaling involved in ECM repair is related to the occurrence of BRONJ.
Our utilization of a gene-score analysis pipeline allowed us to identify more candidate genes than when using traditional genetic models. Excluding genes without known specific functions (
CDC27 and
TNRC18),
ARID2,
HEBP1,
LTBP1, and
PLVAP were the only significant differences in the cancer group revealed by the gene-score methodology, while
DFFA,
FAM193A, and
VEGFA were the only significant differences in the osteoporosis group. In particular, the
VEGFA gene, which differed significantly in the osteoporosis BRONJ, is a member of previous well known risk gene families in ONJ [
12],
VEGF which is a growth factor that plays an important role in angiogenesis, vasculogenesis, and epithelial cell growth [
42]. It has long been known that
VEFG plays an important role in bone formation and repair [
41], and there has also been a GWAS supporting the hypothesis that the impairment of angiogenesis in the tissue surrounding unnecrotized tissue would be involved in the development of BRONJ [
11]. In addition, the
PLVAP gene, which is involved in the structure of the diaphragm and vascular fenestrations identified in the cancer group, may be a downstream target of
VEGF signaling, and is also an important factor in angiogenesis. Otherwise, the
LTBP1 gene that is related to osteoclast activity, which is involved in bone remodeling and the development of BRONJ, has been newly identified in this study. This gene has been shown to release the active form of TGF-β1 in the ECM [
45], and it plays an important role in osteogenesis and bone resorption. Thus, dysfunction of the
LTBP1 gene might also be implicated in the development of BRONJ. The
HEBP1 gene identified in three BRONJ cancer patients is very interesting as well. This gene codes Heme Binding Protein 1 (HBP1), and heme is a complex of iron and tetrapyrrole protoporphyrin IX, which is in the prosthetic group of hemoproteins that play a key role in oxygen binding and the transportation of compounds such as hemoglobin and myoglobin [
46]. An elevated concentration of free heme can induce pro-oxidant, proinflammatory, and cytotoxic effects that affect different cell types. Heme toxicity plays a major role in the pathogenesis of hemolytic disorders such as sickle-cell disease. Only a few studies have investigated the effects of dysfunction of the
HEBP1 gene on HBP production and metabolism, but heme toxicity and BRONJ present with very similar symptoms, and so further studies are needed into this association.
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