Abstract
目的
探索广泛型侵袭性牙周炎(generalized aggressive periodontitis，GAgP)患者牙根形态异常与骨代谢或牙根发育相关基因多态性的关联。
方法
纳入179例GAgP患者，平均(27.23±5.19)岁，男:女=67 : 112，平均存留牙数(26.80±1.84)颗。采用基于基质辅助激光解吸电离飞行时间质谱技术进行9个与骨代谢和牙根发育相关基因的13个单核苷酸多态性位点(single nucleotide polymorphisms，SNPs)的基因型检测。采用全口根尖片评判牙根形态异常，包括锥根、细长根、冠根比例失调、弯曲根、融合根、后牙根形态异常，分析13个SNPs位点不同基因型根形态异常牙的数量及发生率。
结果
GAgP患者根形态异常牙构成比为14.49%(695/4 798颗)，平均(3.88±3.84)颗。维生素D受体(vitamin D receptor，VDR)基因rs2228570位点的CC、CT、TT基因型患者根形态异常牙数量分别为(4.66±4.10)、(3.71±3.93)和(2.68±2.68)颗，CC基因型和TT基因型之间差异有统计学意义(t=2.62，P=0.01)。降钙素受体(calcitotin receptor，CTR)基因rs2283002位点CC、CT、TT基因型患者根形态异常数分别为(5.02±3.70)、(3.43±3.95)、(3.05±3.12)颗，CC基因型的根形态异常发病率高于CT和TT基因型(87.86% vs. 65.26%和63.64%，P=0.006，adjusted OR=3.71，95%CI:1.45~9.50)。
结论
VDR rs2228570及CTR rs2283002位点可能与广泛型侵袭性牙周炎患者牙根形态异常的发生有关，值得进一步研究。
Keywords: 侵袭性牙周炎, 牙根形态异常, 基因, 单核苷酸多态性
Abstract
Objective
To explore the association between the abnormal root morphology and bone metabolism or root development related gene polymorphism in patients with generalized aggressive periodontitis.
Methods
In the study, 179 patients with generalized aggressive periodontitis were enrolled, with an average age of (27.23±5.19) years, male / female = 67/112. The average number of teeth remaining in the mouth was (26.80±1.84). Thirteen single nucleotide polymorphisms (SNPs) of nine genes which related to bone metabolism and root development were detected by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Root abnormalities were identified using periapical radiographs. The abnormal root morphology included cone-rooted teeth, slender-root teeth, short-rooted teeth, curved-rooted teeth, syncretic-rooted molars, and molar root abnormalities. The number of teeth and incidence of abnormal root morphology in different genotypes of 13 SNPs were analyzed.
Results
The constituent ratio of root with root abnormality in GAgP patients was 14.49%(695/4 798). The average number of teeth with abnormal root morphology in GAgP was (3.88±3.84). The average number of teeth with abnormal root morphology in CC, CT and TT genotypes in vitamin D receptor (VDR) rs2228570 was (4.66±4.10), (3.71±3.93) and (2.68±2.68). There was significant difference between TT genotype and CC genotype (t = 2.62, P =0.01). The average number of root morphological abnormalities in CC, CT and TT genotypes of Calcitotin Receptor (CTR) gene rs2283002 was (5.02±3.70), (3.43±3.95), and (3.05±3.12). The incidence of root morphological abnormalities in CC genotype was higher than that in the patients with CT and TT, and the difference was statistically significant(87.86% vs. 65.26% & 63.64%, P=0.006, adjusted OR =3.71, 95%CI: 1.45-9.50). There was no significant difference in the incidence of abnormal root morphology between CT and TT genotypes.
Conclusion
VDR rs2228570 and CTR rs2283002 may be associated with the occurrence of abnormal root morphology in patients with generalized aggressive periodontitis, which is worthy of further research.
Keywords: Aggressive periodontitis, Root abnormality, Gene, Single nucleotide polymorphisms
广泛型侵袭性牙周炎(generalized aggressive periodontitis, GAgP)因发病年龄早、疾病进展快、具有家族聚集性、早期出现牙齿松动和失牙等备受关注。GAgP是多因素的复杂性疾病, 尽管菌斑生物膜是GAgP的始动因素，但与遗传有关的宿主易感性(如基因多态性)和局部解剖因素(如根形态异常)是影响疾病进展速度和严重程度的重要因素。
GAgP被普遍认为与易感基因多态性有关，许多学者已发现多个基因的单核苷酸多态性(single nucleotide polymorphisms，SNPs)与GAgP的易感性增加有关[1]，如维生素D受体(vitamin D receptor，VDR)基因多态性与GAgP的关系已得到验证[2-3]。
以往系列研究已证实我国GAgP患者特有的牙根形态异常(锥根、细长根、冠根比例失常、弯曲根、融合根)的发生率明显高于慢性牙周炎患者和牙周健康者[4-6]; 根形态异常核心家系研究显示有遗传倾向，遗传度为30%~50%[7]; 根形态异常牙的牙槽骨吸收程度明显大于非根形态异常牙，根形态异常牙数与重度骨吸收牙数成正相关，分析原因是这些牙承受
力的能力降低，导致疾病进展加快[8]。
牙根的发育是口腔上皮组织和间充质相互作用的结果，其发育异常可能与某些基因缺失或变异有关，机体内外的不利因素作用于牙齿硬组织形成期、牙根发生期等阶段时，会形成相应的临床表现[9-11]。以往研究表明很多基因都与牙根的发育有关，包括同源异形盒基因1、同源异形盒基因2、骨形成蛋白基因、基质金属蛋白酶9、转化生长因子β、核心结合因子α1、VDR等[12-16]。基础研究还表明降钙素受体(calcitotin receptor，CTR)影响成牙本质细胞分化，不排除与牙根发育有关[17-18], 但既往多是关于基因是否会阻止牙根形成或导致牙根缺失的研究[16, 19-22]，都是基于细胞或动物实验[23-24]，尚未见GAgP患者牙根形态异常与致病基因的关联研究。
本研究旨在初步分析GAgP患者中牙根形态异常与骨代谢和牙根发育相关基因的关系，探索可能与GAgP患者发生牙根形态异常相关的基因多态性位点，为GAgP患者实施针对性的预防和个性化治疗提供理论依据[25]。
1. 资料与方法
1.1. 研究对象
选择2001年1月至2012年12月就诊于北京大学口腔医院牙周科门诊的179例GAgP患者作为研究对象。GAgP患者纳入标准参照1999年牙周病分类法国际研讨会制定的诊断标准(1999年新分类法):(1)患者年龄小于36岁; (2)快速的牙槽骨破坏和附着丧失，全口至少有6颗牙(至少有3颗为非第一磨牙和切牙)的探诊深度(probing depth，PD)≥5 mm，邻面附着丧失≥3 mm，均经全口根尖片证实有邻面牙槽骨吸收; (3)口内余留牙≥20颗; (4)无正畸治疗史; (5)患者除患牙周炎外全身健康，无糖尿病、心血管疾病和血液疾病等系统性疾病; 女性未怀孕及哺乳。
本研究在开始前已获得北京大学生物医学伦理委员会审查批准(批准号:0313)，所有受试者纳入前均签署知情同意书，完成问卷调查，包括身高、体质量、吸烟史、全身健康状况等。
1.2. 牙周临床检查和影像学检查
所有临床检查均由高年资医生完成，检查内容包括:(1)菌斑指数; (2)除第三磨牙外的每颗牙齿6个位点(近中颊、颊侧中央、远中颊、近中舌、舌侧中央和远中舌)的PD、临床附着丧失(attachment loss，AL); (3)每颗牙近中颊、颊侧中央、远中颊、舌侧4个位点的出血指数(bleeding index，BI)。
所有研究对象均在北京大学口腔医院放射科拍摄全口根尖X线片(采用分角线投照技术，每人14张)，将全口根尖X线片扫描后存入电脑(确保X线片比例不变)。
1.3. GAgP患者骨代谢和牙根发育相关基因SNPs的检测
所有受试者采取空腹前臂静脉血10 mL，放入含有乙二胺四乙酸抗凝剂的真空管中，12 000转离心5 min，留取白细胞层，-70 ℃冻存。采用上海华舜生物工程有限公司DNA提取试剂盒从白细胞中提取总基因组DNA，紫外分光光度计测定浓度和纯度，D260 nm /D280 nm为1.8~2.0，-20 ℃保存。根据近十年的文献报道和GenBank的检索信息[12-16]，共筛选和分析了9个与骨代谢和牙根发育相关基因的13个SNPs位点(表 1)。SNPs基因型鉴定是由上海邃志生物科技有限公司利用美国Sequenom公司的MassARRAY系统完成。为了确保分型结果的准确性，对其中3个样本39个位点的SNPs分型进行了重复检测。
表 1.
纳入研究的9个基因及13个SNPs位点
13 SNPs from 9 candidate genes
| Gene | SNPs |
| VDR，vitamin D receptor; CTR，calcitotin receptor; MMP-8, matrix metallopeptidase 8; RANK, activator of NF-κB; DBP, vitamin D binding protein; EGF, epidermal growth factor; BMP2, bone morphogenetic protein-2; S100A8, calprotectin S100A8; Runx2/Cbfa1, core binding factor α1; SNPs, single nucleotide polymorphisms. | |
| VDR | rs2228570 |
| CTR | rs2283002，rs2374634 |
| MMP-8 | rs11225395 |
| RANK | rs11664594 |
| DBP | rs17467825，rs4588，rs7041 |
| EGF | rs2237051 |
| BMP2 | rs2273073 |
| S100A8 | rs3795391, rs3806232 |
| Runx2/Cbfa1 | rs6938177 |
1.4. 牙根形态异常的评估
参照徐莉等[6]、LV等[25]的研究，根据X线片表现将牙根形态异常分为6类:(1)锥根:牙根从根颈1/3即变窄，平面观似三角形，牙根细且短，常见于前牙和双尖牙。当根宽度参照值满足以下条件时，可判断为椎根:上中切牙 < 0.67;上侧切牙 < 0.50;下切牙 < 0.34;上前磨牙 < 0.39;下前磨牙 < 0.42(图 1A、B)。(2)细长根:牙根细，呈杆状，多见于下前牙(图 1C)。(3)冠根比例失调:冠长和根长的比值不同于正常牙。当冠根比满足以下条件时，可判断为冠根比例失调:上中切牙>0.81;上侧切牙>0.90;下切牙>0.80;前磨牙>0.80(图 1D)。(4)弯曲根:前牙和双尖牙可见牙根呈明显弯曲，根尖弯曲部与牙长轴呈15°角以上(图 1E)。(5)融合根:磨牙的牙根融合后似单根牙，多见于上、下颌的第二磨牙(图 1F)。(6)后牙根形态异常:后牙牙根过短或过细(图 1G)。由4位有经验且已经校准的医师在不知患者基因型的(盲法)情况下，阅读X线片，对牙根形态异常状况进行判定。
图 1.
牙根形态异常典型X线片
Typical X-ray films of abnormal root morphology
A, cone-rooted teeth of anterior; B, cone-rooted teeth of premolar; C, slender-root teeth; D, short-rooted teeth; E, curved-rooted teeth; F, syncretic-rooted molars; G, molar root abnormalities.
1.5. 统计学分析
采用SPSS 25.0软件，对患者水平和牙水平进行统计分析。计量资料服从正态分布时以均数±标准差表示，不服从正态分布时以中位数(P25，P75)表示。不同基因型牙根形态异常数量的比较:(1)如果数据服从正态分布且具备方差齐性，采用独立样本t检验; (2)如果数据服从正态分布但不具备方差齐性，采用校正独立样本t检验; (3)如果数据不服从正态分布，则运用非参数检验Mann-Whitney U检验; (4)对多组数据间的比较，如果数据服从正态分布且方差齐，采用单因素方差分析; 如果数据不服从正态分布，则采用Kruskal-Wallis H检验进行分析。不同基因型根形态异常发生率的比较:将同一位点根形态异常发生率差异无统计学意义的两基因型合并为一组后进行多因素Logistic回归分析，计算调整比值比(odds ratio，OR)。双侧P＜0.05时认为差异有统计学意义。
2. 结果
2.1. 一般资料
共纳入179例GAgP患者，其中男:女=67 : 112，平均年龄(27.23±5.19)岁。平均口内存留牙为(26.80±1.84)颗(20~28颗)，平均PD为(4.85±0.98) mm，平均AL为(4.44±1.30) mm，平均BI为3.54±0.45。
2.2. 牙根形态异常总体分布状况
179例GAgP患者口内存留牙共计4 798颗，其中存在牙根形态异常的合计695颗，根形态异常构成比为14.49%，平均(3.88±3.84)颗。在6种类型的牙根形态异常中，前牙和双尖牙锥根较为常见(表 2)。
表 2.
牙根形态异常牙的分布状况
Distribution of teeth with abnormal root morphology
| Item | Teeth level, n (%) |
| Cone-rooted teeth of anterior | 214 (31) |
| Cone-rooted teeth of premolar | 160 (23) |
| Short-rooted teeth | 106 (15) |
| Curved-rooted teeth | 85 (12) |
| Slender-root teeth | 53 (8) |
| Molar root abnormalities | 39 (6) |
| Syncretic-rooted molars | 37 (5) |
| Root abnormalities | 695 (100) |
2.3. 13个SNPs位点不同基因型根形态异常牙分布
在筛选的9个与骨代谢和牙根发育相关基因的13个SNPs位点中，经过统计分析，VDR rs2228570及CTR rs2283002位点不同基因型牙根形态异常数量存在差异，其余SNPs位点不同基因型根形态异常牙的数量及发生率差异无统计意义(表 3、4)。
表 3.
GAgP患者9个基因13个SNPs位点不同基因型与牙根形态异常
Different genotypes of 13 single nucleotide polymorphisms from 9 candidate genes and root morphology abnormalities in patients with GAgP
| SNPs | Gene type | GAgP, n(%) | Cone-rooted teeth of anterior | Teeth of root abnormalities | |||
| Median(P25, P75) | P | x±s/Median(P25, P75) | P | ||||
| VDR, vitamin D receptor; CTR, calcitotin receptor; MMP-8, matrix metallopeptidase 8; RANK, activator of NF-κB; DBP, vitamin D binding protein; EGF, epidermal growth factor; BMP2, bone morphogenetic protein 2; S100A8, calprotectin S100A8; Runx2/Cbfa1, core binding factor α1; SNPs, single nucleotide polymorphisms; GAgP, generalized aggressive periodontitis. | |||||||
| VDR rs2228570 | CC | 59 (33.91) | 0 (0，2) | 4.66±4.10 | |||
| CT | 90 (51.72) | 0 (0，2) | 0.02 | 3.71±3.93 | 0.01 | ||
| TT | 25 (14.37) | 0 (0，0) | 2.68±2.68 | ||||
| CTR rs2283002 | TT | 22 (13.25) | 0 (0，1.25) | 3.05±3.12 | |||
| CT | 95 (57.23) | 0 (0，2) | 0.14 | 3.43±3.95 | 0.03 | ||
| CC | 49 (29.52) | 0 (0，2.50) | 5.02±3.70 | ||||
| CTR rs2374634 | TT | 144 (83.72) | 0 (0，2) | 3 (0，6.75) | |||
| TC | 25 (14.53) | 0 (0，1) | 0.44 | 4 (0，5) | 0.75 | ||
| CC | 3 (1.74) | 0 (0，1) | 6 (0，7) | ||||
| MMP-8 rs11225395 | GG | 73 (41.01) | 0 (0，2) | 4.25±3.95 | |||
| AG | 88 (49.44) | 0 (0，1) | 0.23 | 3.48±3.70 | 0.38 | ||
| AA | 17 (9.55) | 0 (0，3) | 4.41±4.18 | ||||
| RANK rs11664594 | TT | 59 (34.30) | 0 (0，2) | 4.17±4.26 | |||
| AT | 87 (50.58) | 0 (0，2) | 0.80 | 4.18±3.80 | 0.21 | ||
| AA | 26 (15.12) | 0 (0，2) | 2.73±2.86 | ||||
| DBP rs17467825 | AA | 77 (44.77) | 0 (0，2) | 3.84±3.79 | |||
| AG | 73 (42.44) | 0 (0，2) | 0.72 | 3.67±3.62 | 0.45 | ||
| GG | 22 (12.79) | 0 (0，2.50) | 4.86±4.95 | ||||
| DBP rs4588 | CC | 76 (43.18) | 0 (0，2) | 3 (0.25，6) | |||
| CA | 79 (44.89) | 0 (0，2) | 0.51 | 4 (0，6) | 0.93 | ||
| AA | 21 (11.93) | 0 (0，3) | 2 (0，8.50) | ||||
| DBP rs7041 | TT | 97 (55.43) | 0 (0，2) | 3.84±3.60 | |||
| GT | 68 (38.86) | 0 (0，2) | 0.39 | 3.99±4.23 | 0.75 | ||
| GG | 10 (5.71) | 1.5 (0，2.50) | 4.80±3.82 | ||||
| EGF rs2237051 | AA | 73 (41.71) | 0 (0，1) | 3.89±4.06 | |||
| GA | 90 (51.43) | 0 (0，2) | 0.12 | 4.01±3.67 | 0.98 | ||
| GG | 12 (6.86) | 0 (0，2) | 3.83±4.28 | ||||
| BMP2 rs2273073 | TT | 156 (90.17) | 0 (0，2) | 0.81 | 3.96±3.83 | 0.97 | |
| GT | 17 (9.83) | 0 (0，2) | 4.00±4.26 | ||||
| GG | - | - | - | - | - | ||
| S100A8 rs3795391 | TT | 136 (77.27) | 0 (0，2) | 0.76 | 3.91±3.98 | 0.99 | |
| TC | 40 (22.73) | 0 (0，2) | 3.90±3.46 | ||||
| CC | - | - | - | - | - | ||
| S100A8 rs3806232 | TT | 130 (73.03) | 0 (0，2) | 0.41 | 3.79±4.00 | 0.63 | |
| CT | 48 (26.97) | 0 (0，2) | 4.10±3.47 | ||||
| CC | - | - | - | - | - | ||
| Runx2/Cbfa1 | CC | 87 (49.71) | 0 (0，2) | 3.44±3.73 | |||
| rs6938177 | TC | 71 (40.57) | 0 (0，3) | 0.01 | 4.65±4.03 | 0.041 | |
| TT | 17 (9.71) | 0 (0，0.50) | 2.47±2.62 | ||||
表 4.
GAgP患者9个基因13个SNPs位点不同基因型根形态异常发生率
Incidence of root morphological abnormalities in different genotypes of 13 single nucleotide polymorphisms from 9 candidate genes
| SNPs | Gene type | Total | Incidence of root abnormality | |
| n(%) | Adjusted OR(95%CI) | |||
| VDR，vitamin D receptor; CTR，calcitotin receptor; MMP-8, matrix metallopeptidase 8; RANK, activator of NF-κB; DBP, vitamin D binding protein; EGF, epidermal growth factor; BMP2, bone morphogenetic protein 2; S100A8, calprotectin S100A8; Runx2/Cbfa1, core binding factor α1. * P < 0.05, indicate significant differences in the incidence of root abnormality between groups (Logistic regression analysis). | ||||
| VDR rs2228570 | ||||
| CC | 59 | 45 (76.27) | ||
| CT+TT | 115 | 79 (68.70) | 0.93 (0.56, 1.52) | |
| CTR rs2283002 | ||||
| CC | 49 | 43 (87.76) | 3.71 (1.45, 9.50)* | |
| CT+TT | 117 | 76 (64.96) | ||
| CTR rs2374634 | ||||
| TT | 144 | 105 (72.92) | ||
| TC+CC | 28 | 19 (67.86) | 1.28 (0.53, 3.06) | |
| MMP-8 rs11225395 | ||||
| AA | 17 | 12 (70.59) | ||
| GG+AG | 161 | 115 (71.42) | 1.04 (0.35, 3.12) | |
| RANK rs11664594 | ||||
| TT | 59 | 46 (77.93) | ||
| AT+AA | 113 | 75 (66.37) | 1.79 (0.87, 3.72) | |
| DBP rs17467825 | ||||
| GG | 22 | 16 (72.37) | ||
| AA+AG | 150 | 107 (71.33) | 0.93 (0.34, 2.54) | |
| DBP rs4588 | ||||
| AA | 21 | 15 (71.43) | ||
| CA+CC | 155 | 79 (72.26) | 1.04 (0.38, 2.86) | |
| DBP rs7041 | ||||
| TT | 97 | 73 (7526) | ||
| GT+GG | 78 | 54 (69.23) | 1.35 (0.69, 2.63) | |
| EGF rs2237051 | ||||
| AA | 73 | 48 (65.75) | ||
| GA+GG | 102 | 79 (77.45) | 0.56 (0.29, 1.09) | |
| BMP2 rs2273073 | ||||
| TT | 156 | 114 (73.08) | ||
| GT | 17 | 12 (70.59) | 1.13 (0.38, 3.40) | |
| S100A8 rs3795391 | ||||
| TT | 136 | 96 (70.59) | ||
| TC | 40 | 30 (75.00) | 0.80 (0.36, 1.79) | |
| S100A8 rs3806232 | ||||
| TT | 130 | 89 (68.46) | ||
| CT | 48 | 38 (79.17) | 0.57 (0.26, 1.26) | |
| Runx2/Cbfa1 rs6938177 | CC | 87 | 60 (68.97) | |
| TC+TT | 88 | 79 (73.86) | 0.79 (0.41, 1.52) | |
2.3.1. VDR rs2228570位点与牙根形态异常
VDR rs2228570位点CC、CT、TT基因型患者的牙根形态异常数量分别为(4.66±4.10)、(3.71±3.93)、(2.68±2.68)颗。CC基因型患者的牙根形态异常数量显著高于TT基因型者，差异具有统计学意义(P=0.01)。同时该位点CC基因型患者的前牙锥根数量显著高于TT基因型，差异具有统计学意义(P=0.02，表 3)。CC基因型典型根形态异常患者全口根尖片见图 2，CC基因型患者根形态异常发生率高于CT和TT基因型，但差异无统计学意义(表 4)。
图 2.
VDR rs2228570 CC基因型患者根尖片
Periapical radiographs of a GAgP patient with VDR rs2228570 CC genotype
Male, 21 years, VDR rs2228570 CC genotype; Cone-rooted teeth of anterior: 11，21, 32, 31, 41, 42; Curved-rooted teeth: 12, 22; Molar root abnormalities: 36, 46.
2.3.2. CTR rs2283002位点与牙根形态异常
CTR rs2283002 TT、CT、CC基因型患者的牙根形态异常数量分别为(3.05±3.12)、(3.43±3.95)、(5.02±3.70)颗。CC基因型患者的牙根形态异常数量显著高于与TT和CT基因型，差异具有统计学意义(P=0.03，表 3)。22例TT基因型患者中有14例发生根形态异常，95例CT基因型患者中有62例发生根形态异常，49例CC基因型患者中有43例存在根形态异常，根形态异常发生率分别为63.64%、65.26%、87.86%，校正年龄、性别后，CC基因型的根形态异常发生率显著高于CT及CC基因型(adjusted OR=3.71，95%CI:1.45~9.50, 表 4)。
3. 讨论
牙根形态异常在我国GAgP患者中发生率高，是牙周病发生发展的重要危险因素之一。本研究采用分角线投照技术拍摄的X线片来观察牙根形态，与以往研究相同，田雨等[26]的研究比较了在X线片和在离体牙上测得的冠根比例，结果差异无统计学意义，证明使用分角线投照技术观察牙根形态是可靠的。本研究中GAgP患者在牙水平存在根形态异常的比例为14.49%，与以往研究相当[6]。牙根的发育主要是口腔上皮组织和外胚间充质相互作用的结果，某些生物分子在时限、活性强度等方面协调发挥作用，影响牙齿发育，但是目前尚未见牙根形态异常发生的遗传学研究。故本研究根据近十年的文献报道和GenBank的检索信息[12-16]，筛选了与骨代谢和牙根发育相关的9个基因的13个SNPs位点，分析不同基因型牙根形态异常的差异，初步发现与牙根形态异常发生相关的基因位点为VDR rs2228570位点和CTR rs2283002位点。
Berdal等[27]通过免疫细胞化学法观察VDR在大鼠切牙牙胚中的分布, 发现在牙胚发育过程中VDR在所有祖细胞中均有表达, 并在分化过程中逐渐减少, 成釉细胞和成牙本质细胞是1, 25-二羟维生素D3 [(1, 25(OH)2D3]的靶细胞。Papagerakis[28]采用电子显微镜及Northern blot方法研究大鼠，发现釉原蛋白的表达受1, 25(OH)2D3调控，在维生素D(vitamin D，VD)缺乏大鼠切牙牙胚中釉原蛋白表达降低, 而单独注射VD后釉原蛋白表达增加。Onishi[29]也发现1, 25(OH)2D3可通过调节钙结合蛋白的基因表达而影响牙齿的矿化过程。Bailleul-Forestier等[30]采用免疫荧光技术发现在人类牙胚分化的成釉细胞和成牙本质细胞的细胞核可以检测到VDR和钙结合蛋白D28k分布，提示1, 25(OH)2D3可能有助于调节牙釉质和牙本质的形成，这些研究提示VDR可能与牙根发育有关。
VDR是一种配体激活的转录因子，VD与VDR结合可促进基因(如釉原蛋白基因、牙本质磷蛋白基因等)表达，形成各种细胞外基质蛋白，如成釉蛋白、牙本质磷蛋白等，参与牙本质和牙釉质的形成[31]。VDR基因位于12号染色体长臂q13-14区，有11个外显子，rs2228570位点在第二外显子区[32]。rs2228570位点的等位基因C突变为T时，可以产生一个新的起始密码子，导致VDR氨基酸链增加3个氨基酸[33]。尽管Gross等[34]的研究显示该基因位点多态性不影响VDR基因转录以及VDR配体亲和力，但也有研究发现该基因位点突变会致使VDR基因转录活性降低[35-36]。Liu等[37]发现该位点CC型可能会增加侵袭性牙周炎的患病风险(OR=2.45)，以往很多研究也发现该基因型可能会增加广泛型侵袭性牙周炎的患病风险[2, 38]。本研究结果显示, 该位点CT基因型GAgP患者平均根形态异常数量比CC基因型患者平均少0.9颗，TT基因型患者平均根形态异常数量比CC基因型患者平均少2.0颗，两者差异有统计学意义，这表明rs2228570位点的等位基因T可能对阻止牙根形态异常的发生呈弱保护作用。CC基因型可能会导致GAgP根形态异常数量增加，造成牙根解剖形态缺陷，与其他多种因素共同导致GAgP患者严重的牙周组织破坏。
CTR rs2283002位点CC基因型GAgP患者平均根形态异常数量为(5.02±3.70)颗，大于CT基因型和TT基因型患者。此外，发现CC基因型患者的根形态异常发生率高于TT和CT基因型(adjusted OR=3.71，95% CI:1.451~9.500)，表明CTR基因rs2283002位点可能与GAgP患者牙根形态异常有关。降钙素受体基因位于染色体长臂7q21.3，目前发现有11个单核苷酸多态性位点，rs2283002位点位于内含子区域[39-40]。Giroux等[41]发现rs2283002位点基因多态性与下腰椎骨密度有关，但Yanovich等[42]和Xiong等[39]的研究未发现该位点基因多态性与骨密度有关。既往关于降钙素受体基因与牙齿发育关系的研究较少，Mallek等[17]对新生大鼠切牙和磨牙牙胚进行了一项体外实验，体外培养条件分为正常营养组和营养缺乏组，结果显示降钙素能明显抑制正常大鼠牙胚的钙吸收，而增加了营养不良大鼠牙胚的钙吸收，而上述作用是发生在牙胚发育早期。Sakakura等[18]用不同浓度降钙素体外培养小鼠第一磨牙牙胚，发现较低浓度的降钙素能促进成牙本质细胞提前分泌前期牙本质，而较高浓度降钙素则会抑制前期牙本质的形成，这提示当CTR基因表达增强时，降钙素可通过与CTR结合而发挥抑制牙本质形成，从而影响牙根发育。
综上所述，本研究提示VDR rs2228570位点和CTR rs2283002位点可能与GAgP患者牙根形态异常的发生有关，为进一步探究广泛型侵袭性牙周炎患者根形态异常的发生和致病机制打下基础。同时本研究也存在一些局限，在观察牙根形态时使用了分角线投照技术，可能采用平行投照更为准确; 同时本研究缺乏健康对照组，所研究的位点较少，需探索更多与牙根发育可能有关的基因位点。
Contributor Information
孟 焕新 (Huan-xin MENG), Email: kqmeng@126.com.
徐 莉 (Li XU), Email: xulihome@263.net.
References
- 1.Stabholz A, Soskolne WA, Shapira L. Genetic and environmental risk factors for chronic periodontitis and aggressive periodontitis. Periodontology. 2010;53(1):138–153. doi: 10.1111/j.1600-0757.2010.00340.x. [DOI] [PubMed] [Google Scholar]
- 2.Park KS, Nam JH, Choi J. The short vitamin D receptor is associated with increased risk for generalized aggressive periodontitis. J Clin Periodontol. 2006;33(8):524–528. doi: 10.1111/j.1600-051X.2006.00944.x. [DOI] [PubMed] [Google Scholar]
- 3.Li S, Yang MH, Zeng CA, et al. Association of vitamin D receptor gene polymorphisms in Chinese patients with generalized aggressive periodontitis. J Periodontal Res. 2008;43(3):360–363. doi: 10.1111/j.1600-0765.2007.01044.x. [DOI] [PubMed] [Google Scholar]
- 4.McNamara CM, Garvey MT, Winter GB. Root abnormalities, talon cusps, dens invaginati with reduced alveolar bone levels: case report. Int J Paediatr Dent. 1998;8(1):41–45. doi: 10.1046/j.1365-263x.1998.00060.x. [DOI] [PubMed] [Google Scholar]
- 5.梁 鑫. 人类牙根发育异常疾病概述. 中华口腔医学杂志. 2019;54(11):783–787. doi: 10.3760/cma.j.issn.1002-0098.2019.11.012. [DOI] [PubMed] [Google Scholar]
- 6.徐 莉, 孟 焕新, 田 雨, et al. 侵袭性牙周炎患者牙根形态异常的观察. 中华口腔医学杂志. 2009;44(5):266–269. doi: 10.3760/cma.j.issn.1002-0098.2009.05.004. [DOI] [PubMed] [Google Scholar]
- 7.乔 敏, 徐 莉, 孟 焕新, et al. 侵袭性牙周炎核心家系牙槽骨吸收和牙根形态的遗传度分析. 中华口腔医学杂志. 2013;48(10):577–580. doi: 10.3760/cma.j.issn.1002-0098.2013.10.001. [DOI] [PubMed] [Google Scholar]
- 8.孟 焕新, 曹 采方, 和 璐, et al. 临床牙周病学. 北京: 北京大学医学出版社; 2014. pp. 95–99. [Google Scholar]
- 9.Puthiyaveetil JSV, Kota K, Chakkarayan R, et al. Epithelial mesenchymal interactions in tooth development and the significant role of growth factors and genes with emphasis on mesenchyme: a review. J Clin Diagn Res. 2016;10(9):5–9. doi: 10.7860/JCDR/2016/21719.8502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Huang XF, Chai Y. Molecular regulatory mechanism of tooth root development. Int J Oral Sci. 2012;4(4):177–181. doi: 10.1038/ijos.2012.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li JY, Parada G, Yang G. Cellular and molecular mechanisms of tooth root development. Development. 2017;144(3):374–384. doi: 10.1242/dev.137216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Jia SH, Edward KHJ, Lan Y, et al. Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Developmental Biology. 2016;420(1):110–119. doi: 10.1016/j.ydbio.2016.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Vaahtokari A, Aberg T, Thesleff I. Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development. 1996;122(1):121–129. doi: 10.1242/dev.122.1.121. [DOI] [PubMed] [Google Scholar]
- 14.Guo T, Cao G, Liu BY, et al. Cbfα1 hinders autophagy by DSPP upregulation in odontoblast differentiation. Int J Biochem Cell Biol. 2019;115(10):78–89. doi: 10.1016/j.biocel.2019.105578. [DOI] [PubMed] [Google Scholar]
- 15.Balic A, Thesleff I. Tissue interactions regulating tooth development and renewal. Curr Top Dev Biol. 2015;115:157–186. doi: 10.1016/bs.ctdb.2015.07.006. [DOI] [PubMed] [Google Scholar]
- 16.Hanna AE, Sanjad S, Andary R, et al. Tooth development associated with mutations in hereditary vitamin D-resistant rickets. Clin Trans Res. 2018;3(1):28–34. doi: 10.1177/2380084417732510. [DOI] [PubMed] [Google Scholar]
- 17.Mallek HM, Nakamoto T, Nuchtern E, et al. The effect of calcitonin in vitro on tooth germs in protein-energy malnourished rats. J Dent Res. 1979;58(9):1921–1925. doi: 10.1177/00220345790580091901. [DOI] [PubMed] [Google Scholar]
- 18.Sakakura Y, Iida S, Ishizeki K, et al. Ultrastructure of the effects of calcitonin on the development of mouse tooth germs in vitro. Arch Oral Biol. 1984;29(7):507–512. doi: 10.1016/0003-9969(84)90071-2. [DOI] [PubMed] [Google Scholar]
- 19.张 瑞, 黄 晓峰, 张 方明, et al. Nfic在牙根发育中作用的研究. 北京口腔医学. 2013;21(3):121–124. doi: 10.3969/j.issn.1006-673X.2013.03.001. [DOI] [Google Scholar]
- 20.Steele-Perkins G, Butz KG, Lyons GE, et al. Essential role for NFI-C/CTF transcription-replication factor in tooth root development. Mol Cell Biol. 2003;23(3):1075–1084. doi: 10.1128/MCB.23.3.1075-1084.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Huang H, Wang J, Zhang Y, et al. Bone resorption deficiency affects tooth root development in RANKL mutant mice due to attenuated IGF-1 signaling in radicular odontoblasts. Bone. 2018;114:161–171. doi: 10.1016/j.bone.2017.12.026. [DOI] [PubMed] [Google Scholar]
- 22.Zhang R, Yang G, Wu X, et al. Disruption of Wnt/β-catenin signaling in odontoblasts and cementoblasts arrests tooth root development in postnatal mouse teeth. Int J Biol Sci. 2013;9(3):228–236. doi: 10.7150/ijbs.5476. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Chen HM, Guo SY, Xia Y, et al. The role of Rho-GEF Trio in regulating tooth root development through the p38 MAPK pathway. Exp Cell Res. 2018;372(2):158–167. doi: 10.1016/j.yexcr.2018.09.022. [DOI] [PubMed] [Google Scholar]
- 24.张 宇凝, 王 骏周, 陈 晨. 牙根发育调控机制的研究进展. 中华口腔医学杂志. 2020;55(8):591–594. doi: 10.3760/cma.j.cn112144-20191226-00466. [DOI] [PubMed] [Google Scholar]
- 25.LV D, Meng HX, Xu L, et al. Root abnormalities and nonsurgical management of generalized, aggressive periodontitis. J Oral Sci. 2017;59(1):1–8. doi: 10.2334/josnusd.16-0027. [DOI] [PubMed] [Google Scholar]
- 26.田 雨, 徐 莉, 孟 焕新, et al. 单根牙牙根表面积的测量与估算. 北京大学学报(医学版) 2009;44(1):32–35. [Google Scholar]
- 27.Berdal A, Hotton D, Pike JW, et al. Cell- and stage-specific expression of vitamin D receptor and calbindin genes in rat incisor: regulation by 1, 25-dihydroxyvitamin D3. Dev Biol. 1993;155(1):172–179. doi: 10.1006/dbio.1993.1016. [DOI] [PubMed] [Google Scholar]
- 28.Papagerakis P. Differential epithelial and mesenchymal regulation of tooth-specific matrix protein sexpression by 1, 25-dihydroxyvitamin D3 in vivo. Connect Tissue Res. 2002;43(2/3):372–375. doi: 10.1080/03008200290000655. [DOI] [PubMed] [Google Scholar]
- 29.Onishi T. Relationship of vitamin D with calbindin D9k and D28k expression in ameloblasts. Arch Oral Biol. 2008;53(2):117–123. doi: 10.1016/j.archoralbio.2007.09.009. [DOI] [PubMed] [Google Scholar]
- 30.Bailleul-Forestier I, Davideau JL, Papagerakis P, et al. Immunolocalization of vitamin D receptor and calbindin-D28k in human tooth germ. Pediatr Res. 1996;39(4):636–642. doi: 10.1203/00006450-199604000-00013. [DOI] [PubMed] [Google Scholar]
- 31.Botelho J, Machado V, Proença L, et al. Vitamin D deficiency and oral health: a comprehensive review. Nutrients. 2020;12(5):1471–1487. doi: 10.3390/nu12051471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.李 媛媛, 崔 凌凌, 李 鑫, et al. 中国汉族男性原发性痛风与维生素D受体基因rs2228570多态性的遗传易感性研究. 中华内分泌代谢杂志. 2015;31(4):316–319. doi: 10.3760/cma.j.issn.1000-6699.2015.04.007. [DOI] [Google Scholar]
- 33.Gross C, Eccleshall TR, Malloy PJ, et al. The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women. J Bone Miner Res. 1996;11(12):1850–1855. doi: 10.1002/jbmr.5650111204. [DOI] [PubMed] [Google Scholar]
- 34.Gross C, Krishnan AV, Malloy PJ, et al. The vitamin D receptor gene start codon polymorphism: A functional analysis of FokI variants. J Bone Miner Res. 1998;13(11):1691–1699. doi: 10.1359/jbmr.1998.13.11.1691. [DOI] [PubMed] [Google Scholar]
- 35.Egan JB, Thompson PA, Vitanov MV, et al. Vitamin D receptor ligands, adenomatous polyposis coli, and the vitamin D receptor FokI polymorphism collectively modulate beta-catenin activity in colon cancer cells. Mol Carcinogen. 2010;49(4):337–352. doi: 10.1002/mc.20603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Alimirah F, Peng XJ, Murillo G, et al. Functional significance of vitamin D receptor FokI polymorphismin human breast cancer cells. PLoS One. 2011;6(1):e16024. doi: 10.1371/journal.pone.0016024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Liu K, Han B, Meng HX, et al. Influence of rs2228570 on transcriptional activation by the vitamin D receptor in human gingival fibroblasts and periodontal ligament cells. J Clin Periodontol. 2017;88(9):1–19. doi: 10.1902/jop.2017.170030. [DOI] [PubMed] [Google Scholar]
- 38.Li S, Yang MH, Zeng CA, et al. Association of vitamin D receptor gene polymorphisms in Chinese patients with generalized aggressive periodontitis. J Periodontal Res. 2008;43(3):360–363. doi: 10.1111/j.1600-0765.2007.01044.x. [DOI] [PubMed] [Google Scholar]
- 39.Xiong DH, Shen H, Zhao LJ, et al. Robust and comprehensive analysis of 20 osteoporosis candidate genes by very high-density single-nucleotide polymorphism screen among 405 white nuclear families identified significant association and gene-gene interaction. J Bone Miner Res. 2006;21(11):1678–1695. doi: 10.1359/jbmr.060808. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Lawrence AW, Mary EF, Zheng YX, et al. In vitro characterization of a human calcitonin receptor gene polymorphism. Mutat Res Fund Mol M. 2003;522(1/2):93–105. doi: 10.1016/s0027-5107(02)00282-8. [DOI] [PubMed] [Google Scholar]
- 41.Giroux S, Elfassihi L, Clément V, et al. High-density polymorphisms analysis of 23 candidate genes for association with bone mineral density. Bone. 2010;47(5):975–981. doi: 10.1016/j.bone.2010.06.030. [DOI] [PubMed] [Google Scholar]
- 42.Yanovich R, Friedman E, Milgrom R, et al. Candidate gene ana-lysis in israeli soldiers with stress fractures. J Sports Sci Med. 2012;11(1):147–155. [PMC free article] [PubMed] [Google Scholar]