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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (3): 564-570. doi: 10.19723/j.issn.1671-167X.2019.03.028

• 论著 • 上一篇    下一篇

利用二代测序数据探索SPRY基因家族与中国人群非综合征型唇腭裂的关联

周仁1,郑鸿尘1,李文咏1,王梦莹1,王斯悦1,李楠2,李静3,周治波2,吴涛1,朱洪平2△()   

  1. 1. 北京大学公共卫生学院流行病与卫生统计学系,北京 100191
    2. 北京大学口腔医学院·口腔医院,口腔颌面外科 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室 口腔数字医学北京市重点实验室,北京 100081
    3. 北京大学口腔医学院·口腔医院儿童口腔科,北京 100081
  • 收稿日期:2019-03-18 出版日期:2019-06-18 发布日期:2019-06-26
  • 作者简介:朱洪平,医学博士,副主任医师,从事口腔颌面部软组织畸形整形修复,尤其擅长先天性唇腭裂畸形的各项外科治疗和语音评估治疗。2000年至2001年曾至美国匹兹堡大学唇腭裂治疗中心,系统学习了唇腭裂序列治疗和唇腭裂分子遗传学理论。目前在开展唇腭裂治疗的临床研究,同时与北京大学公共卫生学院吴涛教授团队合作,开展唇腭裂畸形病因学研究,在国内外唇腭裂病因学研究领域率先探索以代谢通路为基础的交互作用,对基因组数据深入分析进行了有益尝试,目前课题组已在叶酸/同型半胱氨酸、SUMO及其下游相关基因、烟碱胆碱能受体基因、16p13.3区域相关基因、SPRY家族基因等5个代谢通路或基因家族中发现存在95对显著的基因-基因交互作用。主持北京大学医学部青年科技创新平台发展基金项目1项,作为合作项目负责人,参与国家自然科学基金面上项目和北京市自然科学基金面上项目各1项。曾获北京市科技进步二等奖1项,卫生部科技进步三等奖1项。发表论文30余篇,其中SCI收录8篇,参与编写专著7部。现任中华口腔医学会唇腭裂专业委员会常委、学术秘书,美国微笑列车基金会唇腭裂慈善修复项目医学专家指导委员会秘书,中国人口福利基金会唇腭裂修复项目专家组成员,中国医师协会整形外科分会小儿整形专委会常委,美国腭颅面裂协会会员。
  • 基金资助:
    国家自然科学基金(81573225)和北大医学交叉研究种子基金(BMU2017MX018)-中央高校基本科研业务费

Exploring the association between SPRY gene family and non-syndromic oral clefts among Chinese populations using data of a next-generation sequencing study

Ren ZHOU1,Hong-chen ZHENG1,Wen-yong LI1,Meng-ying WANG1,Si-yue WANG1,Nan LI2,Jing LI3,Zhi-bo ZHOU2,Tao WU1,Hong-ping ZHU2△()   

  1. 1. Department of Epidemiology and Biostatistics, Peking University School of Public Health, Beijing 100191, China
    2. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
    3. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, China
  • Received:2019-03-18 Online:2019-06-18 Published:2019-06-26
  • Supported by:
    Supported by the National Natural Science Foundation of China (81573225) and the Fundamental Research Funds for the Central Universities: Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2017MX018)

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摘要： 目的 探索SPRY基因家族中的单核苷酸多态性及亲源效应与中国人群非综合征型唇腭裂发病风险的关联。方法 研究基于病例-双亲设计,以2016—2018年于北京大学口腔医院募集的 183个中国人群非综合征型唇裂合并或不合并腭裂(non-syndromic cleft lip with or without cleft palate,NSCL/P)核心家系(549人)为研究对象。利用其二代测序数据中的SPRY基因家族相关信息,展开单核苷酸多态性和亲源效应分析。该测序研究采用二阶段设计,第一阶段对24个家系进行全外显子组测序探索潜在阳性信号,第二阶段在159个家系的独立样本中对第一阶段结果进行验证。采用问卷调查收集NSCL/P患者及其父母的基本情况、患病情况、临床特征及孕期环境暴露等信息。收集患儿及其父母的血液并提取DNA进行基因检测以获取遗传信息。单核苷酸多态性分析采用传递不平衡检验,亲源效应分析采用Z检验,统计分析使用PLINK(v1.07)软件完成。第一阶段的多位点分析采用以家系为基础的序列核心关联检验方法,由R软件(v3.5.1)famSKAT-RC包完成。采用Bonferroni法对验证结果进行多重检验校正。结果 经过质量控制,第一阶段共有22个SPRY基因家族的位点纳入分析,结合位点的注释、功能预测结果及统计检验结果,纳入rs1298215244 (SPRY1)和rs504122 (SPRY2)两个位点进入二阶段验证。二阶段传递不平衡检验发现,rs1298215244: T>C、rs504122: G>C两种常见变异以及rs504122: G>T罕见变异与NSCL/P的关联达到Bonferroni多重检验校正后的显著性水平,位于SPRY1的rs1298215244: T>C其亲源效应矫正前具有统计学意义,但未能通过多重检验校正。结论 发现SPRY基因家族中的单核苷酸多态性与中国人群NSCL/P的发病风险存在关联,但未发现SPRY基因家族具有亲源效应。

关键词: 非综合征型唇腭裂, 关联研究, 核心家系, 二代测序技术, SPRY基因家族

Abstract: Objective: To explore the association between SPRY gene family and the risk of non-syndromic oral clefts among Chinese populations, in respect of single nucleotide polymorphisms (SNPs) association and parent-of-origin effects.Methods: Based on case-parent design, this study used the data of SPRY gene family in a next generation sequencing study of 183 non-syndromic cleft lip with or without cleft palate (NSCL/P) case-parent trios (549 participants) recruited from 2016 to 2018, to analyze the effects of SNP association and parent-of-origin. The sequencing study adopted a two-stage design. In the first stage, whole exome sequencing was conducted among 24 NSCL/P trios with family history to explore potential signals. Then in the second stage, another 159 NSCL/P trios were used as validation samples to verify the signals found in the first stage. The data of general information, disease features and parental environmental exposures for participants were collected through questionnaires. Blood samples were collected from each participant for DNA extraction and sequencing. Transmission disequilibrium tests (TDT) were conducted to test for the association between SNPs and NSCL/P, while Z score tests were applied to analyze parent-of-origin effects. The analyses were performed using Plink (v1.07). TRIO package in R (v3.5.1). Besides, famSKAT analyses were conducted in the first stage to combine the effect of SNPs located on the same gene, using famSKAT package in R(V3.5.1). Bonferroni method was adopted to correct multiple tests in the second stage. Results: Twenty-two SNPs in SPRY gene family were included for analyses after the quality control process in the first stage. Based on the variants annotation, functional prediction and statistical analysis, rs1298215244 (SPRY1) and rs504122 (SPRY2) were included in the second verification stage. TDTs in the verification stage revealed that rs1298215244: T>C, rs504122: G>C and rs504122: G>T were associated with the risk of NSCL/P after Bonferroni corrections, where rs504122: G>T was a rare variation. Although the test for parent-of-origin effect of rs1298215244: T>C reached nominal significance level, no SNP showed significant association with NSCL/P through parent-of-origin effect after Bonferroni corrections.Conclusion: This study found that SNPs (including both common and rare variants) among the SPRY gene family were associated with the risk of NSCL/P among Chinese populations. This study failed to detect parent-of-origin effects among the SPRY gene family.

Key words: Non-syndromic oral clefts, Association study, Case-parent trios, Next generation sequencing, SPRY gene family

中图分类号: 

  • R782.2

表1

中国人群183个NSCL/P患者的性别分布情况"

Stage Male Female Total
Stage 1 15 9 24
Stage 2 99 60 159
Total 114 69 183

表2

中国人群24个NSCL/P核心家系SPRY基因家族中单位点的传递不平衡检验结果"

SNP Gene Chromosome Position Number of transmittedallele Number of non-transmitted allele P value
rs300574 SPRY1 4 124323738 18 9 0.083 3
rs4912844 SPRY4 5 141691371 1 5 0.102 5
rs4728 SPRY2 13 80910786 4 8 0.248 2
chr4: 124322600 SPRY1 4 124322600 1 0 0.317 3
rs1440299404 SPRY1 4 124322722 1 0 0.317 3

表3

中国人群24个NSCL/P核心家系中SPRY基因的以家系为基础的序列核心关联检验分析结果"

phi Gene Number of alleles P value
0 SPRY1 5 1
SPRY2 5 0.533 2
SPRY4 12 0.754 2
0.2 SPRY1 5 1
SPRY2 5 0.587 5
SPRY4 12 0.918 3
0.5 SPRY1 5 1
SPRY2 5 0.662 8
SPRY4 12 0.964 0
0.8 SPRY1 5 1
SPRY2 5 0.676 8
SPRY4 12 0.962 2
1 SPRY1 5 1
SPRY2 5 1
SPRY4 12 0.943 6

表4

中国人群159个NSCL/P核心家系中验证位点的传递不平衡检验结果"

Allele Gene Variation MAF OR P value
rs1298215244 SPRY1 T>C 0.114 3.53 4.06×10-6
rs504122 SPRY2 G>C 0.116 2.45 4.81×10-4
rs504122 SPRY2 G>T 0.017 10.00 0.006 7

表5

中国人群159个NSCL/P核心家系中验证位点的亲源效应分析结果"

Allele Gene Variation Paternal transmitted ∶untransmitted counts Maternal transmitted ∶untransmitted counts P value
rs1298215244 SPRY1 T>C 2.5 ∶25.5 12.5 ∶27.5 0.039 6
rs504122 SPRY2 G>C 11 ∶28 9 ∶21 0.870 6
rs504122 SPRY2 G>T 1 ∶3 0 ∶7 -
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