Bisphosphonate related osteonecrosis of the jaw is associated with polymorphisms of the cytochrome P450 CYP2C8 in MM

We have explored the potential role of genetics in the development of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients under bisphosphonate therapy. A genome wide association study was performed using 500.568 single nucleotide polymorphisms (SNPs) in two series of homogeneously treated MM patients: one with ONJ (22 MM cases) and another without ONJ (65 matched MM controls). Four SNPs (rs1934951, rs1934980, rs1341162 and rs17110453) mapped within the Cytochrome P450-2C gene (CYP2C8) showed a different distribution between cases and controls with statistically significant differences (p=1.07x10-6, p=4.231x10-6, p=6.22x10-6 and p=2.15x10-5, respectively). SNP rs1934951 was significantly associated with a higher risk of ONJ development even after Bonferroni correction (P corrected value=0.02). Genotyping results displayed an overrepresentation of the T allele in cases as compared with controls (48% vs. 12%). Thus, individuals homozygous for the T allele had an increased likelihood of developing ONJ (Odds ratio 12.75, 95% confidence interval 3.7 to 43.5). We studied 22 cases (MM with ONJ) and 65 controls (MM without ONJ), matched for age, gender and ethnicity (note: all of the cases and controls are Caucasian). All patients were enrolled in the GEM-00 protocol, which consists on polychemotherapy and autologous transplantation. All received BPs therapy, either Pamidronate (16 cases, 57 controls) or Zoledronic Acid (6 cases, 8 controls) planned for 2 years (median 22 months, range 9-24 months). Clinical characteristics were similar between controls and cases. Study protocols were approved by the ethics committee and written informed consent was obtained from all participants. Each patient was genotyped using the Affymetrix GeneChip Mapping 500K set of microarrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s recommendations. Genotypes were determined using the BRLMM algorithm with cases and controls undergoing joint cluster analysis, after ensuring a robust association test through quality filtering tests. Based on stringent quality control criteria a total of 339.972 SNPs were selected for subsequent analyses. Criteria for exclusion were: 1) call rate<90%, 2) minor allele frequency <5% and 3) deviations from Hardy-Weinberg equilibrium with a p<0.00001. Sexual chromosomes were excluded for analysis. To test for allelic associations between SNPs and ONJ, we constructed 2x2 contingency tables and compared using the two-sided Fisher’s exact or Chisquare tests through SPSS software (SPSS 14.0, Inc. Chicago, IL, USA). P-values were corrected (Pc) using the Bonferroni correction. The strength of association was estimated by the odds ratio (OR), and their 95% confidence intervals (CI) were calculated by Cornfield methods. Linkage disequilibrium between SNPs was analyzed using the Arlequin Software (http://anthro.unige.ch/arlequin).

本研究探讨了双膦酸盐（bisphosphonate）治疗下的多发性骨髓瘤（multiple myeloma, MM）患者发生颌骨骨坏死（osteonecrosis of the jaw, ONJ）的遗传学潜在作用。我们采用500,568个单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs），对两队列接受同质化治疗的多发性骨髓瘤患者开展全基因组关联研究（Genome Wide Association Study）：一组为颌骨骨坏死患者（22例多发性骨髓瘤病例），另一组为无颌骨骨坏死的匹配对照（65例多发性骨髓瘤对照）。定位在细胞色素P450-2C基因（Cytochrome P450-2C, CYP2C8）内的4个单核苷酸多态性位点（rs1934951、rs1934980、rs1341162和rs17110453）在病例组与对照组间的分布存在统计学显著性差异（对应P值分别为1.07×10^-6、4.231×10^-6、6.22×10^-6和2.15×10^-5）。即使经邦弗朗尼校正（Bonferroni Correction）后，单核苷酸多态性位点rs1934951仍与颌骨骨坏死的更高发病风险显著相关（校正后P值=0.02）。基因分型结果显示，病例组中T等位基因的携带率显著高于对照组（48% vs. 12%）。因此，携带T等位基因纯合子的个体发生颌骨骨坏死的风险升高（比值比（Odds Ratio, OR）=12.75，95%置信区间（Confidence Interval, CI）为3.7~43.5）。本研究纳入22例合并颌骨骨坏死的多发性骨髓瘤病例与65例无颌骨骨坏死的多发性骨髓瘤对照，两组在年龄、性别及种族方面均匹配（注：所有研究对象均为高加索人）。所有患者均入组GEM-00方案，该方案包含多药化疗（polychemotherapy）与自体造血干细胞移植（autologous transplantation）；所有患者均接受双膦酸盐治疗，其中帕米膦酸二钠（Pamidronate）组16例病例、57例对照，唑来膦酸（Zoledronic Acid）组6例病例、8例对照，计划治疗时长为2年（中位时长22个月，范围9~24个月）。病例组与对照组的临床特征无显著差异。本研究方案经伦理委员会批准，所有研究对象均签署书面知情同意书。所有患者均按照制造商说明书，采用Affymetrix GeneChip Mapping 500K芯片微阵列（Affymetrix GeneChip Mapping 500K set of microarrays，美国加利福尼亚州圣克拉拉市Affymetrix公司）进行基因分型。基因型检测采用BRLMM算法（BRLMM algorithm），通过联合聚类分析对病例组与对照组进行基因分型；在通过质量过滤测试确保关联检验的可靠性后，基于严格的质量控制标准，最终共筛选出339,972个单核苷酸多态性位点用于后续分析。排除标准如下：1）检出率<90%；2）次要等位基因频率<5%；3）不符合哈迪-温伯格平衡（Hardy-Weinberg equilibrium）且P<0.00001。本研究未分析性染色体上的位点。为检验单核苷酸多态性与颌骨骨坏死之间的等位基因关联，我们构建了2×2列联表，并通过SPSS软件（SPSS 14.0，美国伊利诺伊州芝加哥市SPSS公司）采用双侧Fisher确切概率法（Fisher’s exact test）或卡方检验进行比较。P值采用邦弗朗尼校正得到校正P值（Pc）。关联强度通过比值比（OR）进行估计，其95%置信区间（CI）采用Cornfield法（Cornfield method）计算。单核苷酸多态性之间的连锁不平衡（Linkage Disequilibrium）分析采用Arlequin软件（http://anthro.unige.ch/arlequin）。