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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (3): 387-393. doi: 10.19723/j.issn.1671-167X.2022.03.001

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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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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

CLC Number: 

  • R181.3+3

Table 1

Enrichment of de novo mutations for each functional class"

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

Table 2

Assessing the number of genes with multiple de novo mutations"

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

Table 3

Genes with de novo mutations significantly more than expected"

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

Figure 1

Protein-protein interaction network analysis in STRING database"

Table 4

Scores from the STRING protein-protein analysis restricted to Homo sapiens"

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

Table 5

De novo mutations in the genes interacting with NSCL/P genes"

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
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