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北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (3): 387-393. doi: 10.19723/j.issn.1671-167X.2022.03.001

• 论著 •    下一篇

基于核心家系全外显子组测序数据探索新生突变与非综合征型唇腭裂的关联

陈曦1,王斯悦1,薛恩慈1,王雪珩1,彭和香1,范梦1,王梦莹1,武轶群1,秦雪英1,李劲1,吴涛1,*(),朱洪平2,李静3,周治波2,陈大方1,胡永华1   

  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，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，口腔数字医学北京市重点实验室，国家卫生健康委员会口腔医学计算机应用工程技术研究中心，国家药品监督管理局口腔生物材料重点实验室，北京 100081
    3. 北京大学口腔医学院·口腔医院儿童口腔科，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，口腔数字医学北京市重点实验室，国家卫生健康委员会口腔医学计算机应用工程技术研究中心，国家药品监督管理局口腔生物材料重点实验室，北京 100081
  • 收稿日期:2022-02-24 出版日期:2022-06-18 发布日期:2022-06-14
  • 通讯作者: 吴涛 E-mail:twu@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81573225);国家自然科学基金(81102178);北京市自然科学基金(7172115)

Exploring the association between de novo mutations and non-syndromic cleft lip with or without palate based on whole exome sequencing of case-parent trios

Xi CHEN1,Si-yue WANG1,En-ci XUE1,Xue-heng WANG1,He-xiang PENG1,Meng FAN1,Meng-ying WANG1,Yi-qun WU1,Xue-ying QIN1,Jing LI1,Tao WU1,*(),Hong-ping ZHU2,Jing LI3,Zhi-bo ZHOU2,Da-fang CHEN1,Yong-hua HU1   

  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 Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
    3. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
  • Received:2022-02-24 Online:2022-06-18 Published:2022-06-14
  • Contact: Tao WU E-mail:twu@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81573225);the National Natural Science Foundation of China(81102178);the Natural Science Foundation of Beijing(7172115)

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摘要：

目的: 在中国人非综合征型唇裂伴或不伴腭裂(non-syndromic cleft lip with or without palate, NSCL/P)核心家系中，利用全外显子组测序探索与NSCL/P发病相关的新生突变位点。方法: 对22个中国NSCL/P核心家系进行全外显子组测序，采用基因组分析工具包(Genome Analysis ToolKit, GATK)通过对比亲代与子代同一位点的等位基因识别新生突变位点，采用SnpEff软件对位点进行功能注释。对新生突变位点进行富集分析，检验全外显子区域内存在的新生突变数量是否高于预期值，以及是否存在包含新生突变数量显著高于预期值的基因。通过查阅文献总结既往研究提示与NSCL/P发病存在较强证据支持的基因，根据注释信息筛选能够引起蛋白质改变的新生突变位点，对该类位点所在基因编码的蛋白质与NSCL/P相关基因编码的蛋白质进行交互作用分析。利用R软件的denovolyzeR包进行富集分析(Bonferroni多重检验校正：P=0.05/n，n为基因个数)。利用STRING数据库预测新生突变所在基因与已知NSCL/P致病基因编码的蛋白质间的交互作用。结果: 全外显子组测序得到的位点中共有339 908个位点通过质量控制，经GATK软件比对共筛选出345个高置信度新生突变，其中错义突变44个，无义突变1个，经典剪接位点2个，同义突变20个，内含子区或基因间区位点278个。富集分析显示，全外显子组中引起蛋白质改变的新生突变数量显著高于预期值(P < 0.05)，KRTCAP2、HMCN2、ANKRD36C、ADGRL2和DIPK2A 5个基因所含的新生突变位点高于预期(P < 0.05/(2×19 618))。蛋白质交互作用分析纳入46个包含能够引起蛋白质序列改变的新生突变所在的基因及13个既往研究提示与NSCL/P存在关联的基因，两类基因编码的蛋白质之间存在6组交互作用，其中RGPD4与SUMO1编码的蛋白质的交互作用证据可信度最高，STRING数据库交互作用评分为0.868。结论: 研究为NSCL/P的发病提供了新的证据，对携带新生突变的基因进行功能分析有助于揭示复杂疾病的遗传结构。

关键词: 新生突变, 富集分析, 蛋白质交互作用, 非综合征型唇裂伴或不伴腭裂

Abstract:

Objective: To explore the association between de novo mutations (DNM) and non-syndromic cleft lip with or without palate (NSCL/P) using case-parent trio design. Methods: Whole-exome sequencing was conducted for twenty-two NSCL/P trios and Genome Analysis ToolKit (GATK) was used to identify DNM by comparing the alleles of the cases and their parents. Information of predictable functions was annotated to the locus with SnpEff. Enrichment analysis for DNM was conducted to test the difference between the actual number and the expected number of DNM, and to explore whether there were genes with more DNM than expected. NSCL/P-related genes indicated by previous studies with solid evidence were selected by literature reviewing. Protein-protein interactions analysis was conducted among the genes with protein-altering DNM and NSCL/P-related genes. R package "denovolyzeR" was used for the enrichment analysis (Bonferroni correction: P=0.05/n, n is the number of genes in the whole genome range). Protein-protein interactions among genes with DNM and genes with solid evidence on the risk factors of NSCL/P were predicted depending on the information provided by STRING database. Results: A total of 339 908 SNPs were qualified for the subsequent analysis after quality control. The number of high confident DNM identified by GATK was 345. Among those DNM, forty-four DNM were missense mutations, one DNM was nonsense mutation, two DNM were splicing site mutations, twenty DNM were synonymous mutations and others were located in intron or intergenic regions. The results of enrichment analysis showed that the number of protein-altering DNM on the exome regions was larger than expected (P < 0.05), and five genes (KRTCAP2, HMCN2, ANKRD36C, ADGRL2 and DIPK2A) had more DNM than expected (P < 0.05/(2×19 618)). Protein-protein interaction analysis was conducted among forty-six genes with protein-altering DNM and thirteen genes associated with NSCL/P selected by literature reviewing. Six pairs of interactions occurred between the genes with DNM and known NSCL/P-related genes. The score measuring the confidence level of the predicted interaction between RGPD4 and SUMO1 was 0.868, which was higher than the scores for other pairs of genes. Conclusion: Our study provided novel insights into the development of NSCL/P and demonstrated that functional analyses of genes carrying DNM were warranted to understand the genetic architecture of complex diseases.

Key words: De novo mutations, Enrichment analysis, Protein-protein interactions, Non-syndromic cleft lip with or without palate

中图分类号: 

  • R181.3+3

表1

各功能类别新生突变富集度"

Class Observed Expected Enrichment P
Synonymous variants 18 6.2 2.92 < 0.001
Protein-altering variants 42 15.8 2.66 0.328
  Missense variants 40 13.9 2.89 < 0.001
  Loss-of-function variants 2 1.9 1.04 0.573
    Nonsense variants 1 0.7 1.41 0.508
    Canonical splice site variants 1 0.3 3.05 0.279
All 60 21.9 2.73 < 0.001

表2

包含多个新生突变的基因数量"

Class Obs expMean expMax P n
Synonymous variants 0 0.0 2 1.000 20
Protein-altering variants 1 0.1 2 0.108 47
  Missense variants 1 0.1 2 0.098 44
  Loss-of-function variants 0 0.0 1 1.000 3
    Nonsense variants 0 0.0 0 1.000 1
    Canonical splice site variants 0 0.0 1 1.000 2
All 5 0.2 4 < 0.001 67

表3

存在显著富集新生突变的基因"

Gene Lof_observed Lof_expected Lof_pvalue Prot_observed Prot_expected Prot_pvalue
ADGRL2 NA NA NA 1 0 0.000
ANKRD36C NA NA NA 2 0 0.000
DIPK2A NA NA NA 1 0 0.000
HMCN2 1 0 0.000 1 0 0.000

图1

STRING数据库蛋白质交互作用网络分析"

表4

STRING数据库蛋白质交互作用得分"

Protein Interaction Total score Co-expression Experiment Database Text-mining
MUC5B TP63 0.473 0.000 0.000 0.000 0.473
PPM1J SUMO1 0.668 0.000 0.379 0.000 0.488
RGPD4 SUMO1 0.868 0.064 0.613 0.600 0.202
GSC BMP4 0.523 0.000 0.000 0.000 0.523
PRSS3 BMP4 0.436 0.000 0.000 0.000 0.436
SLIT1 PAX1 0.577 0.000 0.000 0.000 0.577

表5

与NSCL/P存在交互作用的基因中的新生突变"

Position ID Alleles AC MAFa Individual ID Effect Gene
chr1:113257689 - C>T 1 - 0207603A Missense PPM1J
chr2:108487966 rs832357 A>G 6 0.061(489/8030) 0235703A Missense RGPD4
chr9:33795603 rs751787967 G>C 3 0.039(237/6140) 0207603A Missense PRSS3
chr10:98819233 rs1295794649 G>A 2 0.000(0/9046) 227403 Missense SLIT1
chr11:1281880 rs61734162 C>T 2 0.000(0/9544) 202403 Missense MUC5B
chr14:95235318 rs1470361138 G>A 1 0.000(0/7158) 209603 Missense GSC
1 Worley ML , Patel KG , Kilpatrick LA . Cleft lip and palate[J]. Clin Perinatol, 2018, 45 (4): 661- 678.
doi: 10.1016/j.clp.2018.07.006
2 Beaty TH , Murray JC , Marazita ML , et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4[J]. Nat Genet, 2010, 42 (6): 525- 529.
doi: 10.1038/ng.580
3 Nasreddine G, El Hajj J, Ghassibe-Sabbagh M. Orofacial clefts embryology, classification, epidemiology, and genetics[J/OL]. Mutat Res Rev Mutat Res, 2021, 787: 108373(2021-02-28)[2022-02-01]. https://pubmed.ncbi.nlm.nih.gov/34083042/.
4 van Rooij IA , Ludwig KU , Welzenbach J , et al. Non-syndromic cleft lip with or without cleft palate: Genome-wide association study in Europeans identifies a suggestive risk locus at 16p12.1 and supports as a clefting susceptibility gene[J]. Genes (Basel), 2019, 10 (12): 1023.
doi: 10.3390/genes10121023
5 Bishop MR , Diaz Perez KK , Sun M , et al. Genome-wide enrichment of de novo coding mutations in orofacial cleft trios[J]. Am J Hum Genet, 2020, 107 (1): 124- 136.
doi: 10.1016/j.ajhg.2020.05.018
6 Jin ZB , Li Z , Liu Z , et al. Identification of de novo germline mutations and causal genes for sporadic diseases using trio-based whole-exome/genome sequencing[J]. Biol Rev Camb Philos Soc, 2018, 93 (2): 1014- 1031.
doi: 10.1111/brv.12383
7 Conrad DF , Keebler JE , DePristo MA , et al. Variation in genome-wide mutation rates within and between human families[J]. Nat Genet, 2011, 43 (7): 712- 714.
doi: 10.1038/ng.862
8 Veltman JA , Brunner HG . De novo mutations in human genetic disease[J]. Nat Rev Genet, 2012, 13 (8): 565- 575.
doi: 10.1038/nrg3241
9 Coe BP , Stessman HAF , Sulovari A , et al. Neurodevelopmental disease genes implicated by de novo mutation and copy number variation morbidity[J]. Nat Genet, 2019, 51 (1): 106- 116.
doi: 10.1038/s41588-018-0288-4
10 Mitra I , Huang B , Mousavi N , et al. Patterns of de novo tandem repeat mutations and their role in autism[J]. Nature, 2021, 589 (7841): 246- 250.
doi: 10.1038/s41586-020-03078-7
11 Jin SC , Homsy J , Zaidi S , et al. Contribution of rare inherited and de novo variants in 2 871 congenital heart disease probands[J]. Nat Genet, 2017, 49 (11): 1593- 1601.
doi: 10.1038/ng.3970
12 Watkins WS , Hernandez EJ , Wesolowski S , et al. De novo and recessive forms of congenital heart disease have distinct genetic and phenotypic landscapes[J]. Nat Commun, 2019, 10 (1): 4722.
doi: 10.1038/s41467-019-12582-y
13 Ware JS, Samocha KE, Homsy J, et al. Interpreting de novo variation in human disease using denovolyzeR[J/OL]. Curr Protoc Hum Genet, 2015, 87: 7.25.1 -7.25.15(2015-08-06)[2022-02-01]. https://pubmed.ncbi.nlm.nih.gov/26439716/.
14 Szklarczyk D , Gable AL , Nastou KC , et al. The STRING database in 2021:Customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets[J]. Nucleic Acids Res, 2021, 49 (D1): D605- D612.
doi: 10.1093/nar/gkaa1074
15 Saleem K , Zaib T , Sun W , et al. Assessment of candidate genes and genetic heterogeneity in human non syndromic orofacial clefts specifically non syndromic cleft lip with or without palate[J]. Heliyon, 2019, 5 (12): e03019.
doi: 10.1016/j.heliyon.2019.e03019
16 Vezain M , Lecuyer M , Rubio M , et al. A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis[J]. Acta Neuropathol Commun, 2018, 6 (1): 109.
doi: 10.1186/s40478-018-0610-5
17 Shao R , Liu J , Yan G , et al. Cdh1 regulates craniofacial development via APC-dependent ubiquitination and activation of Goosecoid[J]. Cell Res, 2016, 26 (6): 699- 712.
doi: 10.1038/cr.2016.51
18 Hamann J , Aust G , Araç D , et al. International union of basic and clinical pharmacology. XCIV. Adhesion G protein-coupled receptors[J]. Pharmacol Rev, 2015, 67 (2): 338- 367.
doi: 10.1124/pr.114.009647
19 Sevastre AS , Buzatu IM , Baloi C , et al. ELTD1:An emerging silent actor in cancer drama play[J]. Int J Mol Sci, 2021, 22 (10): 5151.
doi: 10.3390/ijms22105151
20 Lek M , Karczewski KJ , Minikel EV , et al. Analysis of protein-coding genetic variation in 60, 706 humans[J]. Nature, 2016, 536 (7616): 285- 291.
doi: 10.1038/nature19057
21 Huang N , Lee I , Marcotte EM , et al. Characterising and predicting haploinsufficiency in the human genome[J]. PLoS Genet, 2010, 6 (10): e1001154.
doi: 10.1371/journal.pgen.1001154
22 Hiramatsu H , Tadokoro S , Nakanishi M , et al. Latrotoxin-induced exocytosis in mast cells transfected with latrophilin[J]. Toxicon, 2010, 56 (8): 1372- 1380.
doi: 10.1016/j.toxicon.2010.08.002
23 Zepeda-Mendoza CJ , Bardon A , Kammin T , et al. Phenotypic interpretation of complex chromosomal rearrangements informed by nucleotide-level resolution and structural organization of chromatin[J]. Eur J Hum Genet, 2018, 26 (3): 374- 381.
doi: 10.1038/s41431-017-0068-0
24 Passi GR , Bhatnagar S . Rhombencephalosynapsis[J]. Pediatr Neurol, 2015, 52 (6): 651- 652.
doi: 10.1016/j.pediatrneurol.2015.02.005
25 Birnbaum S , Ludwig KU , Reutter H , et al. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24[J]. Nat Genet, 2009, 41 (4): 473- 477.
doi: 10.1038/ng.333
26 Mangold E , Ludwig KU , Birnbaum S , et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate[J]. Nat Genet, 2010, 42 (1): 24- 26.
doi: 10.1038/ng.506
27 Beaty TH , Taub MA , Scott AF , et al. Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study[J]. Hum Genet, 2013, 132 (7): 771- 781.
doi: 10.1007/s00439-013-1283-6
28 Parry DA , Logan CV , Stegmann AP , et al. SAMS, a syndrome of short stature, auditory-canal atresia, mandibular hypoplasia, and skeletal abnormalities is a unique neurocristopathy caused by mutations in Goosecoid[J]. Am J Hum Genet, 2013, 93 (6): 1135- 1142.
doi: 10.1016/j.ajhg.2013.10.027
29 Ulmer B , Tingler M , Kurz S , et al. A novel role of the organizer gene Goosecoid as an inhibitor of Wnt/PCP-mediated convergent extension in Xenopus and mouse[J]. Sci Rep, 2017, 7, 43010.
doi: 10.1038/srep43010
30 Yu Y , Zuo X , He M , et al. Genome-wide analyses of non-syndromic cleft lip with palate identify 14 novel loci and genetic heterogeneity[J]. Nat Commun, 2017, 8, 14364.
doi: 10.1038/ncomms14364
31 Kalisz M , Winzi M , Bisgaard HC , et al. EVEN-SKIPPED HOMEOBOX 1 controls human ES cell differentiation by directly repressing GOOSECOID expression[J]. Dev Biol, 2012, 362 (1): 94- 103.
doi: 10.1016/j.ydbio.2011.11.017
32 Rivera-Pérez JA , Mallo M , Gendron-Maguire M , et al. Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development[J]. Development, 1995, 121 (9): 3005- 3012.
doi: 10.1242/dev.121.9.3005
33 Yamada G , Mansouri A , Torres M , et al. Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death[J]. Development, 1995, 121 (9): 2917- 2922.
doi: 10.1242/dev.121.9.2917
34 Feitosa NM , Zhang J , Carney TJ , et al. Hemicentin 2 and fibulin 1 are required for epidermal-dermal junction formation and fin mesenchymal cell migration during zebrafish development[J]. Dev Biol, 2012, 369 (2): 235- 248.
doi: 10.1016/j.ydbio.2012.06.023
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ISSN 1671-167X
CN 11-4691/R
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