儿童氟斑牙与钙代谢相关基因多态性的关系
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摘要
地方性氟中毒的病因早已明确,但有研究发现,同一氟接触水平的人群中,其个体患病与否及患病程度存在差异,提示遗传因素可能在氟致机体损伤中起一定作用。
目的：
探讨高氟暴露区儿童氟斑牙遗传易感性与ER、VDR基因多态性的关系,为进一步探讨高氟暴露条件下基因-环境相互作用提供基础资料。
对象与方法：
1.调查点、调查对象的选取：在水氟监测基础上,选取河南省开封、通许两县作为调查点,两点各包括1个高氟区(水氟平均浓度>2.0mg/L)和1个对照区(水氟平均浓度<1.0mg/L)。高氟区与对照区的其他条件等均相似。整群抽取两地8～12岁儿童进行健康体检,并排除影响钙磷代谢的因素后,对符合调查要求的237名儿童进行氟斑牙检查,并据此分为3组：氟斑牙患者组、高氟区对照组、非病区对照组。全部对象均知情、同意。
2.氟斑牙诊断：由口腔执业医师及公共卫生执业医师按Dean's分级法诊断。
3.标志物检测：原子分光光度计法测定血清钙；放射免疫法测定血清骨钙素及血清降钙素；氟离子选择电极法检测尿氟。
4.基因多态性分析：利用PCR-RFLP技术。
5.数据统计分析：采用SPSS 12.0软件。主要方法有：两独立样本的t检验、单因素方差分析、列联表资料的χ2检验、多因素非条件Logistic回归等,以a=0.05为检验水准。
结果：
1.儿童氟斑牙的环境流行病学调查：高氟区氟斑牙患病率为51.7%。氟斑牙患者组的年龄大于高氟区对照组,二者差异有统计学意义(P<0.05)。
2.儿童氟斑牙与ER基因多态性的关系：
(1) ER RsaⅠ等位基因频率在高氟区患者组与非患者组的差异有统计学意义(OR=1.821,95% CI:1.013-3.274)。
(2) ER PvuⅡ各基因型及等位基因频率的分布差异均无统计学意义(均为P>0.05)。
(3)在尿氟超标的氟斑牙患者组和高氟区对照组儿童中,等位基因频率差异有统计学意义(OR=0.535,95%CI:0.305-0.941)。
(4) ER RsaⅠ、ER PvuⅡ、ER XbaⅠ多态性在不同性别氟斑牙患者中分布差异无统计学意义(P>0.05)。
3.儿童氟斑牙与VDR基因多态性的关系：VDR FokⅠ各基因型及等位基因频率的分布差异均无统计学意义(均为P>0.05)；各基因型在不同性别氟斑牙患者中的分布差异无统计学意义(P>0.05)。
结论：
1.儿童氟斑牙与高氟区儿童ER RsaⅠ多态性可能有相关性。与携带有野生r等位基因的儿童相比,携带有突变R等位基因的儿童患氟斑牙的危险性更高。
2.在尿氟超标的儿童中,携带ER XbaⅠ位点X等位基因可能降低氟斑牙发生的危险性。
3.儿童氟斑牙与高氟区儿童ER PvuⅡ、VDR FokⅠ多态性可能无相关性。
The mechanisms of endemic fluorosis have been well studied in the past decades. Researchers found that human body can be affected by fluoride in different levels such as tissue, cell, and molecular level and so on. However, not all the people suffer from fluorosis when exposed to high fluoride. It suggested that genetic susceptibility may play a role in endemic fluorosis.
Objective:
In the current study, we analyzed the variation in ER and VDR genotypes in dental fluorosis cases and control children to investigate the relationship between children's dental fluorosis and VDR,ER gene polymorphisms.
Meterials and Methods:
1.Selection of investigation area and samples
A pilot study was conducted in four villages of two counties (Kaifeng and Tongxu) in Henan Province. Both counties include one endemic fluorosis village (EFV) and one non-endemic fluorosis village (NEFV). The average levels of fluoride in drinking water exceeded 2.0 mg/L in EFV. The average levels of fluoride in drinking water was less than 1.0 mg/L in NEFV. In addition to different fluoride concentration in drinking water, both EFV and NEFV have the similar levels of nature condition, economy condition, construction of population, calcium in the diet and the similar levels of calcium ion and magnesium ion in drinking water. Children aged 8 to 12 years old, born and raised in the four villages were recruited excluding those who have received drug treatment in the form of bisphosphonates, calcitonin, fluoride, or hormone replacement therapy and/or who had hip fractures. A total of 237 children participated in this study. All participants were examined for DF using the Dean's Method and divided into 3 groups:cases, controls form EFV, and controls form NEFV. All procedures were approved by the Institutional Review Board at Zhengzhou University, China (IRB00006861, FWA00014064).
2. Children's dental fluorosis inspection
Dentist and public health practitioners diagnosed dental fluorosis in accordance with the Dean's classified method.
3 Detection of biomarkers
Serum calcium levels was measured by using flame atomic absorption spectrometry. Serum osteoealcin and calcitonin level were inspect by using radioimmunoassay. The urine fluoride and water fluoride levels in samples were detected by using fluoride ion selective electrode.
4 Genotype analysis
The amplification of target fragments by using PCR method and the detection of polymorphisms of ER gene RsaⅠ,PvuⅡ,XbaⅠand VDR gene Fok I by using restricted fragment length polymorphisms method. 5 Data analysis
All analyses were performed using the SPSS software, version 12.0. Major methods used in the resarch included chi-square test, analysis of variance, T test, multivariate logistic regression and Hardy-Weinberg Equilibrium test and so on. All tests of significance were two-sided. A P-value of less than 0.05 was considered statistically significant.
Results:
1 Distributions of select variables in dental fluorosis cases and controls
There were 74 subjects who were diagnosed with dental fluorosis. All of them lived in EFV, the prevalence of dental fluorosis was 51.7%(74/143). There were nearly 89.2%(66/74) of cases whose urine fluoride levels exceeding 1.5mg/L.The exceeded rate of urine fluoride was 84.6%(55/69) and 9.6%(9/94) in controls of EFV and NEFV respectively. The dental fluorosis cases were older than controls from EFV (P<0.05).
2 The relationship between ER gene and dental fluorosis
2.1 The frequency distribution of ERβRsaⅠgenotype was rr 60.8%(45/74), Rr 27.0%(20/74), RR 12.1%(9/74) in children with dental fluorosis, rr 73.9%(51/69), Rr 20.2%(14/69), RR 5.8%(4/69) in children without dental fluorosis from EFV, and rr 63.8%(60/94), Rr 34.0%(32/94), RR 2.1%(2/94) in children from NEFV respectively. Children carrying r allele of ERβRsal has a significantly increased risk of dental fluorosis(OR= 1.821,95% CI:1.013-3.274) compared to children carrying R allele of ERβRsaⅠin EFV. No significant difference was found in allele frequency of ERβRsaⅠgenotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05).
2.2 The frequency distribution of ER PvuⅡgenotype was PP20.3%(15/74), Pp36.5%(27/74), pp42.7%(32/74) in children with dental fluorosis, PP11.6%(8/69), Pp46.4%(32/69), pp42%(29/69) in children without dental fluorosis from EFV, PP23.4%(22/94), Pp 47.9%(45/94), pp28.7%(27/94) in the children without fluorosis from NEFV respectively. No Significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of ER Pvu II genotypes between cases and non-dental fluorosis in high-loaded fluoride status (P>0.05)
2.3 The frequency distribution of ER Xba I genotype was XX6.8%(5/74), Xx 36.5%(27/74), xx 56.8%(42/74) in children with dental fluorosis, XX15.9%(11/69), Xx37.7%(26/69), xx46.4%(32/69), in children without dental fluorosis from EFV XX14.9%(14/94), Xx43.6%(41/94), xx 41.5%(39/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). Significant difference was found in allele frequency of ER Xba I genotypes between cases and non-dental fluorosis when children with high-loaded fluoride status (OR=0.535,95%CI:0.305-0.941).
2.4 No statistical difference was found in distribution of RsaⅠ,PvuⅡ,XbaⅠgenotypes of ER gene in boys and girls with dental fluorosis (P>0.05).
3 The relationship between VDR gene and dental fluorosis
The frequency distribution of VDR Fok I genotype was FF32.4%(24/74), Ff 45.9% (34/74),ff21.6%(16/74) in children with dental fluorosis, FF40.6%(28/69), Ff36.2% (25/69), ff23.2%(16/69) in children without dental fluorosis from EFV, FF 31.9%(30/94), Ff50.0%(47/94),ff18.1%(17/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of VDR Fok I genotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05). No statistical difference was found in distribution of Fok I polymorphism of VDR gene in boys and girls with dental fluorosis (P>0.05).
Conclusion:
1 The ER gene RsaⅠpolymorphism may be associated with the risk of dental fluorosis in a high fluoride exposed populations.
2 Children who carried X allele frequency of ER XbaⅠhad a lower risk of dental fluorosis when children with high-loaded fluoride status.
3 There was no correlation between ER PvuⅡand VDR FokⅠpolymorphism and dental fluorosis.
目的：
探讨高氟暴露区儿童氟斑牙遗传易感性与ER、VDR基因多态性的关系,为进一步探讨高氟暴露条件下基因-环境相互作用提供基础资料。
对象与方法：
1.调查点、调查对象的选取：在水氟监测基础上,选取河南省开封、通许两县作为调查点,两点各包括1个高氟区(水氟平均浓度>2.0mg/L)和1个对照区(水氟平均浓度<1.0mg/L)。高氟区与对照区的其他条件等均相似。整群抽取两地8～12岁儿童进行健康体检,并排除影响钙磷代谢的因素后,对符合调查要求的237名儿童进行氟斑牙检查,并据此分为3组：氟斑牙患者组、高氟区对照组、非病区对照组。全部对象均知情、同意。
2.氟斑牙诊断：由口腔执业医师及公共卫生执业医师按Dean's分级法诊断。
3.标志物检测：原子分光光度计法测定血清钙；放射免疫法测定血清骨钙素及血清降钙素；氟离子选择电极法检测尿氟。
4.基因多态性分析：利用PCR-RFLP技术。
5.数据统计分析：采用SPSS 12.0软件。主要方法有：两独立样本的t检验、单因素方差分析、列联表资料的χ2检验、多因素非条件Logistic回归等,以a=0.05为检验水准。
结果：
1.儿童氟斑牙的环境流行病学调查：高氟区氟斑牙患病率为51.7%。氟斑牙患者组的年龄大于高氟区对照组,二者差异有统计学意义(P<0.05)。
2.儿童氟斑牙与ER基因多态性的关系：
(1) ER RsaⅠ等位基因频率在高氟区患者组与非患者组的差异有统计学意义(OR=1.821,95% CI:1.013-3.274)。
(2) ER PvuⅡ各基因型及等位基因频率的分布差异均无统计学意义(均为P>0.05)。
(3)在尿氟超标的氟斑牙患者组和高氟区对照组儿童中,等位基因频率差异有统计学意义(OR=0.535,95%CI:0.305-0.941)。
(4) ER RsaⅠ、ER PvuⅡ、ER XbaⅠ多态性在不同性别氟斑牙患者中分布差异无统计学意义(P>0.05)。
3.儿童氟斑牙与VDR基因多态性的关系：VDR FokⅠ各基因型及等位基因频率的分布差异均无统计学意义(均为P>0.05)；各基因型在不同性别氟斑牙患者中的分布差异无统计学意义(P>0.05)。
结论：
1.儿童氟斑牙与高氟区儿童ER RsaⅠ多态性可能有相关性。与携带有野生r等位基因的儿童相比,携带有突变R等位基因的儿童患氟斑牙的危险性更高。
2.在尿氟超标的儿童中,携带ER XbaⅠ位点X等位基因可能降低氟斑牙发生的危险性。
3.儿童氟斑牙与高氟区儿童ER PvuⅡ、VDR FokⅠ多态性可能无相关性。
The mechanisms of endemic fluorosis have been well studied in the past decades. Researchers found that human body can be affected by fluoride in different levels such as tissue, cell, and molecular level and so on. However, not all the people suffer from fluorosis when exposed to high fluoride. It suggested that genetic susceptibility may play a role in endemic fluorosis.
Objective:
In the current study, we analyzed the variation in ER and VDR genotypes in dental fluorosis cases and control children to investigate the relationship between children's dental fluorosis and VDR,ER gene polymorphisms.
Meterials and Methods:
1.Selection of investigation area and samples
A pilot study was conducted in four villages of two counties (Kaifeng and Tongxu) in Henan Province. Both counties include one endemic fluorosis village (EFV) and one non-endemic fluorosis village (NEFV). The average levels of fluoride in drinking water exceeded 2.0 mg/L in EFV. The average levels of fluoride in drinking water was less than 1.0 mg/L in NEFV. In addition to different fluoride concentration in drinking water, both EFV and NEFV have the similar levels of nature condition, economy condition, construction of population, calcium in the diet and the similar levels of calcium ion and magnesium ion in drinking water. Children aged 8 to 12 years old, born and raised in the four villages were recruited excluding those who have received drug treatment in the form of bisphosphonates, calcitonin, fluoride, or hormone replacement therapy and/or who had hip fractures. A total of 237 children participated in this study. All participants were examined for DF using the Dean's Method and divided into 3 groups:cases, controls form EFV, and controls form NEFV. All procedures were approved by the Institutional Review Board at Zhengzhou University, China (IRB00006861, FWA00014064).
2. Children's dental fluorosis inspection
Dentist and public health practitioners diagnosed dental fluorosis in accordance with the Dean's classified method.
3 Detection of biomarkers
Serum calcium levels was measured by using flame atomic absorption spectrometry. Serum osteoealcin and calcitonin level were inspect by using radioimmunoassay. The urine fluoride and water fluoride levels in samples were detected by using fluoride ion selective electrode.
4 Genotype analysis
The amplification of target fragments by using PCR method and the detection of polymorphisms of ER gene RsaⅠ,PvuⅡ,XbaⅠand VDR gene Fok I by using restricted fragment length polymorphisms method. 5 Data analysis
All analyses were performed using the SPSS software, version 12.0. Major methods used in the resarch included chi-square test, analysis of variance, T test, multivariate logistic regression and Hardy-Weinberg Equilibrium test and so on. All tests of significance were two-sided. A P-value of less than 0.05 was considered statistically significant.
Results:
1 Distributions of select variables in dental fluorosis cases and controls
There were 74 subjects who were diagnosed with dental fluorosis. All of them lived in EFV, the prevalence of dental fluorosis was 51.7%(74/143). There were nearly 89.2%(66/74) of cases whose urine fluoride levels exceeding 1.5mg/L.The exceeded rate of urine fluoride was 84.6%(55/69) and 9.6%(9/94) in controls of EFV and NEFV respectively. The dental fluorosis cases were older than controls from EFV (P<0.05).
2 The relationship between ER gene and dental fluorosis
2.1 The frequency distribution of ERβRsaⅠgenotype was rr 60.8%(45/74), Rr 27.0%(20/74), RR 12.1%(9/74) in children with dental fluorosis, rr 73.9%(51/69), Rr 20.2%(14/69), RR 5.8%(4/69) in children without dental fluorosis from EFV, and rr 63.8%(60/94), Rr 34.0%(32/94), RR 2.1%(2/94) in children from NEFV respectively. Children carrying r allele of ERβRsal has a significantly increased risk of dental fluorosis(OR= 1.821,95% CI:1.013-3.274) compared to children carrying R allele of ERβRsaⅠin EFV. No significant difference was found in allele frequency of ERβRsaⅠgenotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05).
2.2 The frequency distribution of ER PvuⅡgenotype was PP20.3%(15/74), Pp36.5%(27/74), pp42.7%(32/74) in children with dental fluorosis, PP11.6%(8/69), Pp46.4%(32/69), pp42%(29/69) in children without dental fluorosis from EFV, PP23.4%(22/94), Pp 47.9%(45/94), pp28.7%(27/94) in the children without fluorosis from NEFV respectively. No Significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of ER Pvu II genotypes between cases and non-dental fluorosis in high-loaded fluoride status (P>0.05)
2.3 The frequency distribution of ER Xba I genotype was XX6.8%(5/74), Xx 36.5%(27/74), xx 56.8%(42/74) in children with dental fluorosis, XX15.9%(11/69), Xx37.7%(26/69), xx46.4%(32/69), in children without dental fluorosis from EFV XX14.9%(14/94), Xx43.6%(41/94), xx 41.5%(39/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). Significant difference was found in allele frequency of ER Xba I genotypes between cases and non-dental fluorosis when children with high-loaded fluoride status (OR=0.535,95%CI:0.305-0.941).
2.4 No statistical difference was found in distribution of RsaⅠ,PvuⅡ,XbaⅠgenotypes of ER gene in boys and girls with dental fluorosis (P>0.05).
3 The relationship between VDR gene and dental fluorosis
The frequency distribution of VDR Fok I genotype was FF32.4%(24/74), Ff 45.9% (34/74),ff21.6%(16/74) in children with dental fluorosis, FF40.6%(28/69), Ff36.2% (25/69), ff23.2%(16/69) in children without dental fluorosis from EFV, FF 31.9%(30/94), Ff50.0%(47/94),ff18.1%(17/94) in children from NEFV respectively. No significant difference was found among the three groups (P>0.05). No significant difference was found in allele frequency of VDR Fok I genotypes between cases and non-dental fluorosis in high-loaded fluoride status(P>0.05). No statistical difference was found in distribution of Fok I polymorphism of VDR gene in boys and girls with dental fluorosis (P>0.05).
Conclusion:
1 The ER gene RsaⅠpolymorphism may be associated with the risk of dental fluorosis in a high fluoride exposed populations.
2 Children who carried X allele frequency of ER XbaⅠhad a lower risk of dental fluorosis when children with high-loaded fluoride status.
3 There was no correlation between ER PvuⅡand VDR FokⅠpolymorphism and dental fluorosis.
引文
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