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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (3): 556-563. doi: 10.19723/j.issn.1671-167X.2019.03.027

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Analysis of single-nucleotide polymorphism of Sonic hedgehog signaling pathway in non-syndromic cleft lip and/or palate in the Chinese population

Jie-ni ZHANG1,*,Feng-qi SONG1,*,Shao-nan ZHOU1,Hui ZHENG1,Li-ying PENG1,Qian ZHANG2,Wang-hong ZHAO3,Tao-wen ZHANG4,Wei-ran LI1,Zhi-bo ZHOU5,Jiu-xiang LIN1△(),Feng CHEN2△()   

  1. 1. Department of Orthodontics, 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
    2. Department of Center Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China
    3. Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510000, China
    4. Department of Orthodontics, Yantai Stomatological Hospital, Yantai 264000, Shandong, China
    5. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China
  • Received:2019-03-20 Online:2019-05-10 Published:2019-06-26
  • Contact: Jie-ni ZHANG,Feng-qi SONG E-mail:jxlin@pku.edu.cn;chenfeng2011@hsc.pku.edu.cn
  • Supported by:
    Supported by the Fundamental Research Funds for the Central Universities: Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2018MX017), the Fundamental Research Funds for the Central Universities: Peking University Medicine Fund of Fostering Young Scholars’ Scientific & Technological Innovation (BMU2018PY025), and the National Natural Science Foundation(81870747,81860194)

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Abstract: Objective: To study the relationship between Sonic hedgehog (Shh) associated single-nucleotide polymorphism (SNP) and non-syndromic cleft lip and/or palate (NSCL/P), and to explore the risk factors of cleft lip and/or palate. Many studies suggest that the pathogenesis of NSCL/P could be related to genes that control early development, in which the Shh signaling pathway plays an important role.Methods: Peripheral blood was collected from 197 individuals (100 patients with NSCL/P and 97 healthy controls). Haploview software was used for haplotype analysis and Tag SNP were selected, based on the population data of Han Chinese in Beijing of the international human genome haplotype mapping project. A total of 27 SNP were selected for the 4 candidate genes of SHH, PTCH1, SMO and GLI2 in the Shh signaling pathway. The genotypes of 27 SNP were detected and analyzed by Sequenom mass spectrometry. The data were analyzed by chi-squared test and an unconditional Logistic regression model. Results: The selected SNP basically covered the potential functional SNP of the target genes, and its minimum allele frequency (MAF) was >0.05: GLI2 73.5%, PTCH1 91.0%, SMO 100.0%, and SHH 75.0%. It was found that the genotype frequency of SNP (rs12674259) located in SMO gene and SNP (rs2066836) located in PTCH1 gene were significantly different between the NSCL/P group and the control group. Linkage disequilibrium was also found on 3 chromosomes (chromosomes 2, 7 and 9) where the 4 candidate genes were located. However, in the analysis of linkage imbalance haplotype, there was no significant difference between the disease group and the control group.Conclusion: In China, NSCL/P is the most common congenital disease in orofacial region. However, as it is a multigenic disease and could be affected by multiple factors, such as the external environment, the etiology of NSCL/P has not been clearly defined. This study indicates that Shh signaling pathway is involved in the occurrence of NSCL/P, and some special SNP of key genes in this pathway are related to cleft lip and/or palate, which provides a new direction for the etiology research of NSCL/P and may provide help for the early screening and risk prediction of NSCL/P.

Key words: Non-syndromic cleft lip and/or palate, Sonic hedgehog, Single-nucleotide polymorphism, Sequennom massarray

CLC Number: 

  • R393

"

Table 2

Tests of HWE for all SNP"

CHR SNP A1 A2 GENO(A1A1/A1A2/A2A2) P
2 rs17390009 G C 0/4/67 1.000
2 rs7604538 T C 19/46/31 0.836
2 rs735557 A G 11/42/42 1.000
2 rs4848122 T C 13/40/32 1.000
2 rs7582470 A G 23/40/34 0.104
2 rs1992900 C T 14/48/33 0.674
2 rs895479 G A 3/39/55 0.266
2 rs4848124 C T 3/39/53 0.261
2 rs277555 C T 4/40/52 0.414
2 rs277536 A G 13/39/42 0.493
2 rs1187935 C A 7/31/48 0.577
2 rs3738880 A C 21/52/23 0.540
7 rs2718107 A C 22/38/35 0.093
7 rs4731562 A G 8/43/43 0.638
7 rs2566871 T C 24/41/32 0.154
7 rs12674259 T G 4/19/77 0.062
7 rs9607 A G 5/37/52 0.786
7 rs4728160 G C 3/37/55 0.385
7 rs1233560 C T 1/40/56 0.038
7 rs208684 C A 4/33/58 1.000
9 rs16909859 A G 5/22/73 0.069
9 rs357564 G A 21/39/33 0.204
9 rs2236407 G A 12/42/43 0.821
9 rs2066836 T C 0/15/75 1.000
9 rs2277184 G A 2/20/75 0.632
9 rs2297088 A G 13/42/42 0.657
9 rs2282041 G A 1/22/74 1.000

Table 3

Chi-squared analysis of allele in controls and NSCL/P patients"

SNP A1 A2 CHISQ P OR SE 95%CI
rs17390009 G C 0.605 0.437 1.736 0.718 0.425-7.086
rs7604538 T C 0.044 0.833 0.956 0.214 0.629-1.454
rs735557 A G 0.138 0.710 0.919 0.227 0.589-1.434
rs4848122 T C 0.869 0.351 1.256 0.245 0.777-2.031
2 rs4848124 C T 0.256 0.4776
2 rs277555 C T 0.254 0.3475
2 rs277536 A G 0.342 0.435
2 rs1187935 C A 0.260 0.4666
2 rs3738880 A C 0.490 0.4396
7 rs2718107 A C 0.418 0.4318
7 rs4731562 A G 0.338 0.3347
7 rs2566871 T C 0.447 0.4588
7 rs12674259 T G 0.160 0.0426
7 rs9607 A G 0.244 0.266
7 rs4728160 G C 0.235 0.2468
7 rs1233560 C T 0.239 0.4629
7 rs208684 C A 0.236 0.2683
9 rs16909859 A G 0.178 0.1479
9 rs357564 G A 0.414 0.3814
9 rs2236407 G A 0.343 0.3832
9 rs2066836 T C 0.090 0.1094
9 rs2277184 G A 0.150 0.0865
9 rs2297088 A G 0.360 0.4098
9 rs2282041 G A 0.113 0.1163

Table 4

Logistic tests of association in controls and NSCL/P patients"

CHR SNP A1 A2 OR SE 95%CI P
2 rs17390009 G C 2.173 0.746 0.503-9.380 0.298
2 rs17390009 G C 2.173 0.746 0.503-9.380 0.298
2 rs7604538 T C 0.960 0.212 0.634-1.453 0.846
2 rs735557 A G 0.902 0.224 0.582-1.398 0.644
2 rs4848122 T C 1.266 0.243 0.787-2.037 0.331
2 rs7582470 A G 1.041 0.202 0.701-1.545 0.843
2 rs1992900 C T 1.146 0.230 0.731-1.796 0.553
2 rs895479 G A 0.947 0.259 0.570-1.573 0.834
2 rs4848124 C T 1.387 0.261 0.832-2.312 0.209
2 rs277555 C T 1.125 0.254 0.684-1.850 0.644
2 rs277536 A G 0.974 0.227 0.625-1.518 0.907
2 rs1187935 C A 0.975 0.247 0.601-1.583 0.920
2 rs3738880 A C 1.004 0.209 0.666-1.513 0.986
7 rs2718107 A C 0.882 0.209 0.586-1.329 0.549
7 rs4731562 A G 1.374 0.245 0.850-2.221 0.195
7 rs2566871 T C 0.888 0.207 0.592-1.333 0.567
7 rs12674259 T G 0.696 0.298 0.388-1.249 0.225
7 rs9607 A G 0.861 0.253 0.525-1.413 0.553
7 rs4728160 G C 1.003 0.255 0.608-1.653 0.991
7 rs1233560 C T 1.450 0.271 0.852-2.466 0.171
7 rs208684 C A 1.206 0.247 0.744-1.955 0.447
9 rs16909859 A G 0.752 0.251 0.460-1.231 0.257
9 rs357564 G A 0.871 0.207 0.581-1.308 0.507
9 rs2236407 G A 0.981 0.227 0.629-1.529 0.931
9 rs2066836 T C 1.224 0.358 0.607-2.467 0.572
9 rs2277184 G A 1.475 0.297 0.825-2.637 0.190
9 rs2297088 A G 1.043 0.215 0.684-1.589 0.846
9 rs2282041 G A 0.666 0.346 0.338-1.313 0.241

Table 5

Association statistics of the NSCL/P and control panels"

SNP Genotype Control NSCL/P OR (95%CI) P(OR) P(Logistic) P(Bonferroni) P(HWE)
rs12674259 G/G 77 (77%) 61 (62.9%) 1
G/T 19 (19%) 36 (37.1%) 0.42 (0.22-0.81) 0.0017 0.0016 0.043 0.062
T/T 4 (4%) 0 (0%) NA (0.00-NA)
rs7582470 A G 0.062 0.804 1.054 0.213 0.695-1.599
rs1992900 C T 0.337 0.561 1.134 0.217 0.742-1.735
rs895479 G A 0.088 0.767 0.930 0.247 0.573-1.507
rs4848124 C T 1.851 0.174 1.402 0.249 0.861-2.283
rs277555 C T 0.375 0.540 1.162 0.246 0.718-1.882
rs277536 A G 0.000 0.989 1.003 0.225 0.646-1.559
rs1187935 C A 0.001 0.974 0.992 0.253 0.604-1.628
rs3738880 A C 0.000 1.000 1.000 0.211 0.661-1.512
rs2718107 A C 0.347 0.556 0.881 0.215 0.578-1.343
rs4731562 A G 1.596 0.207 1.336 0.229 0.852-2.093
rs2566871 T C 0.304 0.581 0.890 0.212 0.587-1.348
rs12674259 T G 1.527 0.217 0.698 0.292 0.393-1.237
rs9607 A G 0.450 0.503 0.846 0.250 0.519-1.380
rs4728160 G C 0.001 0.973 0.992 0.250 0.607-1.619
rs1233560 C T 1.640 0.200 1.380 0.252 0.842-2.261
rs208684 C A 0.790 0.374 1.248 0.250 0.765-2.035
rs16909859 A G 1.489 0.222 0.714 0.277 0.415-1.228
rs357564 G A 0.479 0.489 0.860 0.218 0.561-1.318
rs2236407 G A 0.002 0.965 1.010 0.221 0.655-1.558
rs2066836 T C 0.310 0.578 1.239 0.386 0.581-2.642
rs2277184 G A 2.396 0.122 1.588 0.300 0.881-2.859
rs2297088 A G 0.114 0.736 1.077 0.219 0.701-1.653
rs2282041 G A 1.227 0.268 0.686 0.342 0.351-1.340

Figure 1

Distribution frequency and multiplex polymerase chain reaction"

Figure 2

Linkage disequilibrium blocks in the GLI2(2q14), SMO(7q32), SHH(7q36) and PTCH1(9q22)"

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