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中华关节外科杂志(电子版)

中华关节外科杂志(电子版) ›› 2020, Vol. 14 ›› Issue (04) : 454 -460. doi: 10.3877/cma.j.issn.1674-134X.2020.04.011

所属专题： 文献；

基础论著

膝骨关节炎与相关金属蛋白酶的基因多态性关联研究
高山1, 孙乾坤2, 王敬威3, 胡梅4, 郭艳幸3,†()   
  1. 1. 261041　潍坊市中医院颈腰痛科
    2. 471000　洛阳市第一中医院急诊科
    3. 471000　洛阳正骨医院（河南省骨科医院）
    4. 410208　长沙，湖南中医药大学
  • 收稿日期:2019-06-20 出版日期:2020-08-01
  • 通信作者: 郭艳幸
  • 基金资助:
    国家中医药管理局"平乐郭氏正骨流派传承工作室"建设项目([2012]228); 河南省科技厅"国家非物质文化遗产—平乐郭氏正骨术的系统研究与开发"项目(112102310008); 河南省中医药管理局"河南省中医临床学科领军人才培育计划"项目(HNZYLJ20131009)

Association of susceptibility to knee osteoarthritis and related metalloproteinases gene polymorphisms

Shan Gao1, Qiankun Sun2, Jingwei Wang3, Mei Hu4, Yanxing Guo3,†()   

  1. 1. Neck and waist pain Department of Weifang Traditional Chinese Medicine Hospital, Weifang 261041, China
    2. Emergency Department of Luoyang No. 1 Traditional Chinese Medicine Hospital, Luoyang 471000, China
    3. Luoyang Orthopedic Hospital(Henan province Orthopedic Hospital), Luoyang 471000, China
    4. Hunan University of Traditional Chinese Medicine, Changsha 410208, China
  • Received:2019-06-20 Published:2020-08-01
  • Corresponding author: Yanxing Guo
  • About author:
    Corresponding author: Guo Yanxing, Email:
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引用本文：

高山, 孙乾坤, 王敬威, 胡梅, 郭艳幸. 膝骨关节炎与相关金属蛋白酶的基因多态性关联研究[J]. 中华关节外科杂志(电子版), 2020, 14(04): 454-460.

Shan Gao, Qiankun Sun, Jingwei Wang, Mei Hu, Yanxing Guo. Association of susceptibility to knee osteoarthritis and related metalloproteinases gene polymorphisms[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2020, 14(04): 454-460.

目的

探讨带有血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶5(ADAMTS-5)基因单核苷酸多态性(SNP)与膝骨关节炎的关联性。

方法

连续收集洛阳正骨医院188例膝骨关节炎患者作为病例组，对照组收集排除疾病诊断的100人。应用基因组变异服务器(GVS)网站选择ADAMTS-5基因共计15个SNP位点，采用基质辅助激光解析飞行时间质谱分析技术(MALDI-TOF-MS)进行SNP分型，先行哈迪温伯格(HW)平衡检测，对符合HW平衡的SNP位点等位基因进行卡方检验及单体型分析，对基因型进行logistic回归分析。

结果

病例组rs2249350位点C等位基因显著多于对照组[比值比(OR)＝1.176，95%置信区间(CI)(1.025，1.351)，P＝0.016]，A等位基因显著少于对照组[OR＝0.761，95%CI(0.612，0.947)，P＝0.016]；rs2249350位点AA基因型[OR＝0.288，95%CI(0.124，0.669)，P＝0.004]及隐性基因模型[OR＝0.348，95%CI(0.162，0.749)，P＝0.007]均与膝关节骨关节炎负相关；rs229054、rs2249350两位点存在连锁不平衡，构成GC、GA、AC共3个单体型区块，其中病例组单体型GC显著多于对照组[OR＝1.259，95%CI(1.032，1.537)，P＝0.019]，而GA则显著少于对照组[OR＝0.763，95%CI(0.614，0.949)，P＝0.017]。

结论

ADAMTS-5基因rs2249350位点C等位基因可能是膝关节骨关节炎的致病性因素，A等位基因则可能是保护性因素；rs2249350位点AA基因型可能是膝关节骨关节炎的保护性基因型，降低患病风险，且A等位基因可能为隐性基因，隐性遗传；rs229054、rs2249350位点GC单体型可能是膝关节骨关节炎的致病性因素，而GA单体型则可能是保护性因素。该位点等位基因、基因型及单体型与膝关节骨关节炎的关系仍需进一步深入研究和验证。

关键词: 骨关节炎, 膝, ADAMTS-5蛋白, 多态性, 单核苷酸, 单元型, 遗传相关性研究
Objective

To investigate the relationship between knee osteoarthritis (KOA) susceptibility and single nucleotides polymorphism (SNP) of a disinterring and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) gene.

Methods

A total of 188 patients with KOA from Luoyang Orthopedic Hospital were collected as the case group, and 100 patients in the control group were excluded from disease diagnosis. Fifteen SNP of ADAMTS-5 genes were chosen by Genome Variation Server (GVS) online gene database. SNP typing was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The Hardy-Weinberg equilibrium test was carried out first, chi-square test and Haplotype analysis were carried out on the allele of SNP locus in accordance with the Hardy-Weinberg balance, and then logistic regression analysis was carried out on the genotype.

Results

The C allele of rs2249350 locus in the case group was significantly higher than that in the control group [odds ratios (OR)=1.176, 95% confidence interval (CI) (1.025, 1.351), P=0.0016], the A allele was significantly lower than that in the control group [OR=0.761, 95%CI (0.612, 0.947), P=0.016]. AA genotype [OR=0.288, 95%CI (0.124, 0.669), P=0.004] and recessive gene model [OR=0.348, 95%CI (0.162, 0.749), P=0.007] at rs2249350 locus were negatively correlated with osteoarthritis of the knee joint. There was linkage disequilibrium between the two sites of rs229054 and rs2249350, which formed three single haplotype blocks of GC, GA, and AC. The haplotype GC in the case group was significantly higher than that in the control group [OR=1.259, 95%CI (1.032, 1.537), P=0.019], while the GA in the case group was markedly lower than that in the control group [OR=0.763, 95%CI (0.614, 0.949), P=0.017].

Conclusions

The C allele of rs2249350 site of ADAMTS-5 gene may be the pathogenic factor of knee osteoarthritis, while the A allele may be the protective factor. AA genotype at rs2249350 site may be a protective genotype of osteoarthritis of the knee joint, which reduces the risk of disease, and A allele may be a recessive gene and recessive inheritance. GC haplotype at rs229054 and rs2249350 site may be the pathogenic factor of knee osteoarthritis, while GA haplotype may be a protective factor. The relationship between alleles, genotypes and haplotypes of this site and osteoarthritis of the knee joint still needs to be further studied and verified.

Key words: Osteoarthritis, knee, ADAMTS-5 protein, Polymorphism, single nucleotide, Haplotype, Genetic association studies
表1 SNP引物序列
SNP位点 正向引物序列(5'-3') 反向引物序列(5'-3') 单碱基延伸引序列(5'-3')
rs226794 ACGTTGGATGTGATTCAGGTGACCGATGGC ACGTTGGATGAGCCAGTTCTCACACACTTC GGCACTGAATGTAGGC
rs162497 ACGTTGGATGATAGTGGCAGACGTGTTTCC ACGTTGGATGTGGTCACTTTTTTCCCCTTG TTGTTCCCCGGAGCAGTC
rs7510287 ACGTTGGATGTGTATACAGTTTGGGGAAGG ACGTTGGATGTGGATGCCTCTAGAGCTCAC CAGGGAAGGAGTAAAAGGG
rs233894 ACGTTGGATGCTAATTCCAACATCTAGTCC ACGTTGGATGGACCAGTGTGACATAGGTAT ACATCTAGTCCTAAATGAGC
rs2830585 ACGTTGGATGTGCTCCCAGAAACAACGGAC ACGTTGGATGTTGGGAGCAGCGTACCATTG TTTTAAGAGGGCCATCTACC
rs233598 ACGTTGGATGCCTTAGGTGTATCAAGAGAC ACGTTGGATGGCAGAGACTACATTAATTGG CTGTTCATCGTAAAGGTTGGC
rs2830586 ACGTTGGATGAAGTGGAAACTTTGTCACAG ACGTTGGATGGATGAATTGCTTAGCACTGG GGAAACTTTGTCACAGATACAG
rs151065 ACGTTGGATGTGCACAAAGATAACTGGAGG ACGTTGGATGGAAGCATGACTTTCTGTGCG CCCCTAACTGGAGGATTCAGCAC
rs162496 ACGTTGGATGCTGAGGTCTCAATTAGACAG ACGTTGGATGGGGAAAATGCAGATACTGGG GGGTTAAAAGATGTAAACTGCAA
rs162509 ACGTTGGATGCCAAGGTACAGTGTAATTGC ACGTTGGATGGTGTCAGGAACATTAATGCC AATCGCATAATTCACAATATTGCC
rs229054 ACGTTGGATGGCATTGTAAATTTGTTGTGG ACGTTGGATGCAGAGCCCATAATTGCATTC TTTGTTGTGGAATATCATTTTATC
rs162499 ACGTTGGATGACAGAGATGGACCATATTAG ACGTTGGATGTCCTAAGTACTGAGCACACC CCATATTAGAAGACATAATTTACAA
rs2249350 ACGTTGGATGAGCACACTGGAGTCTTTCAC ACGTTGGATGCCATAATTTGCAAACATGCCG GCATTTTATAATTAACCACTATAAAG
rs233896 ACGTTGGATGGTGCACAGAAGGGCATAATC ACGTTGGATGACAACGTAGGAGAAAGGTGG ATAATCATATATCAAAGATTTAGGTTT
rs151058 ACGTTGGATGTCTGGGTATGAAGCTGTTAG ACGTTGGATGATCCTCTATCACAGGGAATC GTGGAGCTGTTAGATGCATTAGGTATA
表2 一般资料比较(例)
一般资料 性别(男/女) 年龄段(中年/年轻老年) BMI区间(体重过低＋正常)/超重/肥胖 职业类别(脑力/体力)
病例组 79/109 82/106 69/74/45 47/141
对照组 54/46 55/45 48/37/15 34/66
χ2值 3.769 3.391 4.648 2.616
P值 >0.05 >0.05 >0.05 >0.05
表3 病例组和对照组SNP等位基因分布情况(例)
SNP rs226794 rs162497 rs7510287 rs2830585 rs233598 rs2830586 rs151065 rs162496 rs229054 rs162499 rs2249350 rs151058
碱基 G/A A/G A/G T/C T/G G/T G/A G/A A/G G/A A/C T/C
病例组 174/194 81/245 30/344 32/344 36/340 34/342 172/200 80/296 63/289 73/283 121/253 41/329
对照组 98/98 37/137 22/178 19/181 25/173 21/179 93/101 35/165 36/162 34/158 85/115 21/177
χ2值 0.378 0.807 1.403 0.158 1.272 0.321 0.148 1.165 0.007 0.621 5.831 0.03
P值 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
表4 KOA与SNP基因型关联性分析结果(例)
SNP 病例组 对照组 OR 95% CI P值 SNP 病例组 对照组 OR 95% CI P值
rs226794 AAa 46 26 1 - - rs151065 AAa 53 27 1 - -
AG 102 46 1.405 (0.693，2.846) 0.345 AG 94 47 1.093 (0.553，2.158) 0.789
GG 36 26 0.776 (0.337，1.790) 0.552 GG 39 23 0.766 (0.335，1.750) 0.526
GG＋AG vs. AAb 138/46 72/26 1.180 (0.605，2.298) 0.627 GG＋AG vs. AAb 133/53 70/27 0.987 (0.518，1.880) 0.968
GG vs. AG＋AAc 36/148 26/72 0.631 (0.310，1.215) 0.161 GG vs. AG＋AAc 39/147 23/74 0.722 (0.361，1.443) 0.356
rs162497 GGa 93 54 1 - - rs162496 AAa 119 69 1 - -
GA 59 59 1.248 (0.651，2.393) 0.505 AG 58 27 1.374 (0.728，2.591) 0.327
AA 11 4 1.789 (0.489，6.542) 0.380 GG 11 4 1.948 (0.539，7.042) 0.309
AA＋GA vs. GGb 70/93 33/54 1.318 (0.707，2.454) 0.385 GG＋AG vs. AAb 69/119 31/69 1.452 (0.797，2.648) 0.223
AA vs. GA＋GGc 11/152 4/83 1.636 (0.459，5.833) 0.448 GG vs. AG＋AAc 11/177 4/96 1.757 (0.494，6.244) 0.384
rs7510287 GGa 158 79 1 - - rs229054 GGa 120 67 1 - -
GA 28 20 0.518 (0.247，1.085) 0.081 GA 49 28 1.154 (0.596，2.232) 0.672
AA 1 1 0.660 (0.018，24.892) 0.823 AA 7 4 1.081 (0.211，5.528) 0.926
AA＋GA vs. GGb 29/158 21/79 0.523 (0.252，1.083) 0.081 AA＋GA vs. GGb 56/120 32/67 1.146 (0.606，2.165) 0.675
AA vs. GA＋GGc 1/186 1/99 0.713 (0.021，24.159) 0.851 AA vs. GA＋GGc 7/169 4/95 1.031 (0.206，5.175) 0.970
rs2830585 CCa 157 82 1 - - rs162499 AAa 116 66 1 - -
CT 30 17 0.856 (0.390，1.877) 0.697 AG 51 26 1.201 (0.627，2.301) 0.582
TT 1 1 0.051 (0.003，0.965) 0.047d GG 11 4 1.899 (0.526，6.853) 0.328
TT＋CT vs. CCb 31/157 18/82 0.776 (0.360，1.673) 0.517 GG＋AG vs. AAb 62/116 30/66 1.298 (0.703，2.397) 0.404
TT vs. CT＋CCc 1/187 1/99 0.053 (0.003，0.997) 0.050 GG vs. AG＋AAc 11/167 4/92 1.791 (0.505，6.360) 0.367
rs233598 GGa 153 76 1 - - rs2249350 CCa 89 37 1 - -
GT 34 21 0.705 (0.338，1.471) 0.352 CA 75 41 0.699 (0.371，1.318) 0.269
TT 1 2 0.046 (0.003，0.827) 0.037d AA 23 22 0.288 (0.124，0.669) 0.004d
TT＋GT vs. GGb 35/153 23/76 0.641 (0.312，1.315) 0.225 AA＋CA vs. CCb 98/89 63/37 0.553 (0.307，0.997) 0.051
TT vs. GT＋GGc 1/187 2/97 0.050 (0.003，0.906) 0.043d AA vs. CA＋CCc 23/164 22/78 0.348 (0.162，0.749) 0.007d
rs2830586 TTa 155 82 1 - - rs151058 CCa 147 79 1 - -
TG 32 15 1.150 (0.518，2.555) 0.731 CT 35 18 3.137 (0.319，30.812) 0.327
GG 1 3 0.029 (0.002，0.377) 0.007d TT 3 2 3.288 (0.315，34.267) 0.320
TT＋GT vs. GGb 33/155 18/82 0.897 (0.422，1.910) 0.779 TT＋CT vs. CCb 38/147 20/79 0.969 (0.466，2.013) 0.932
TT vs. GT＋GGc 1/187 3/97 0.028 (0.002，0.365) 0.006d TT vs. CT＋CCc 3/182 2/97 0.315 (0.032，3.065) 0.320
图1 LD(连锁不平衡)分析单体型区块图
表5 单体型分析结果(例)
编号 单体型 单体型频数 χ2值 P值 OR 95%CI
病例组 对照组
区块1 CTAGAGC 219.5/156.5 118.1/81.9 0.022 0.881 0.989 (0.857，1.142)
CTGAGGC 79.3/296.7 34.8/165.2 1.014 0.314 1.201 (0.838，1.719)
CTAGAAC 30.1/345.9 22.0/178.0 1.451 0.228 0.725 (0.430，1.223)
TGAGAGT 32.0/344.0 19.0/181.0 0.158 0.691 0.896 (0.521，1.539)
CTAGAGT 10.4/365.6 1.9/198.1 1.043 0.307 2.660 (0.588，12.021)
编号 单体型 单体型频数 χ2值 P值 OR 95%CI
病例组 对照组
区块2 GC 186.6/189.4 78.7/121.3 5.502 0.019* 1.259 (1.032，1.537)
GA 121.9/254.1 85.0/115.0 5.731 0.017* 0.763 (0.614，0.949)
AC 67.5/308.5 36.3/163.7 0.000 0.991 1.002 (0.695，1.445)
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