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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2020 Oct 1;13(10):2576–2585.

Association analysis of GWAS hits and non-syndromic cleft lip with/without palate with cleft alveolar in Han population of western China

Shijun Duan 1,*, Jiayu Shi 2,*, Bing Shi 1, Zhonglin Jia 1
PMCID: PMC7642695  PMID: 33165418

Abstract

Cleft alveolar is often accompanied by non-syndromic cleft lip with/without palate (NSCL/P), which could seriously affect the growth and development of the maxilla. In this study, we assessed the associations between 47 susceptible SNPs from previous GWASs of NSCL/P and cleft alveolar in Western Han Chinese population. We recruited 228 trios of NSCL/P with cleft alveolar (156 males and 72 females). The 47 SNPs were genotyped by SNPscan method; Hardy-Weinberg equilibrium test, TDT and parent-of-origin effects were analyzed by PLINK; linkage disequilibrium analysis was conducted by Haploview software. TDT analysis revealed FOXE1 rs894673 (P = 0.0071, ORtransmission = 0.35, 95% CI: 0.16-0.78) and rs3758249 (P = 0.0071, ORtransmission = 0.35, 95% CI: 0.16-0.78) were associated with NSCL/P accompanied cleft alveolar bone. Parent-of-origin effect analysis revealed a paternal special under-transmission of allele A at rs894673 (P = 0.039), allele T at rs3759249 (P = 0.039), and allele T at rs4460498 (P = 0.039) of FOXE1. Allele A at rs987525 showed a significant paternal over-transmission (P = 0.0077). Pairwise LD analysis showed strong LD among rs894673, rs3759249 and rs4460498 (r2 > 0.95, D’ = 1). To conclude, our findings indicated that FOXE1 is the susceptible gene for cleft alveolar accompanied by NSCL/P.

Keywords: Cleft alveolar, non-syndromic cleft lip with/without palate, susceptible gene, association study

Introduction

Non-syndromic cleft lip with or without cleft palate (NSCL/P), as common congenital birth defects, comprise a range of morphological abnormalities of oral and maxillofacial regions. Based on previous epidemiologic studies, Chinese have higher incidences of NSCL/P compared to other ethnicities [1-3], and the prevalence of NSCL/P was more than 1.6% in China [4,5]. In particular, the incidence rate of NSCL/P in western regions was significantly higher than that of other regions of China [5].

Cleft alveolus is a severe bony defect malformation that is present in 75% of NSCL/P [6]. The cleft alveolar refers to a space in the alveolar bone of maxilla with a discontinuity of the dental arch. This bony defect of the alveolus can prevent normal eruption of the cleft-adjacent permanent teeth, disrupt the stability of the maxillary segments, and maintain an oronasal fistula [6-8]. Each of these problems requires a special rehabilitation to avoid malocclusion, maxillary constriction or face asymmetry. Despite the mature sequential therapy involving alveolar bone grafting, the patients had maxillary growth retardation and craniofacial abnormalities thereafter [9].

Achieving a successful and well-functioning reconstruction is the ideal goal of cleft alveolar repair. Alveolar bone grafting is generally considered a gold standard for alveolar cleft treatment. However, the failure rate of this procedure was up to 15%, and normally accompanied with a serious of complications including pain, bleeding, infection, fracture, scar, or chronic pain [8-10]. Bearing in mind all the above-mentioned implications accompanying cleft alveolus repair, the pathogenesis of this deformity is worthwhile to explore.

Genome-wide association study (GWAS) has obvious advantages in the etiology of genetic diseases, and is widely used to unravel the genetic basis of complex diseases [11-13]. Previous GWASs had revealed many susceptibility genes/loci for NSCL/P, which had provided more clues for detecting the etiology of clefts [14-19]. Until now, only 20% of the heritability could be explained. NSCL/P is still an important birth defect affecting quality of the birth in China. Etiological research is crucial to the prevention and control of this disease.

The premaxilla and upper lip come from the maxillary processes and medial nasal process. Cleft alveolus, as a common phenotype of bone malformation, is often accompanied by NSCL/P. Previous GWASs referring to NSCL/P identified susceptible genes of cleft lip. Unfortunately, there are no published data regarding the genetic background of cleft alveolus. In this study, we conducted an experiment with caseparent trios design to validate the association between 47 single nucleotide polymorphisms (SNPs) and cleft alveolus. The purpose of this study was to identify the susceptible genes related to cleft alveolar.

Materials and methods

Ethics statement

All the participants signed the informed consent before being recruited in this study. For patients younger than 16 years old, the informed consents were written by their guardians. The study protocols were reviewed and approved by the Hospital Ethics Committee (HEC) of West China Hospital of Stomatology (No. 2010080), Sichuan University.

Samples description

We recruited 228 case-parent trios of NSCL/P with cleft alveolar, including 156 males and 72 females of the probands, who hospitalized between 2008 and 2013 in the Cleft Lip and Palate Surgery Department of West China Stomatology College, Sichuan University. The probands with non-syndromic cleft lip with or without cleft palate accompanied with cleft alveolus and their parents were enrolled in this study. All the patients were checked by at least 2 professional maxillofacial doctors of the West China Hospital of Stomatology. The probands with other congenital deformities or mental retardation were excluded. All participants were restricted as Western Han Chinese according to self-identification.

SNPs selection and genotyping

Based on the previous GWAS findings about NSCL/P, we selected 47 SNPs with the most significant P values in published studies. Primary information of these SNPs is shown in Table 1.

Table 1.

Primary information on 47 SNPs

Chr SNP Position (Hg19) Gene Function
1 rs4920522 18940380 PAX7 intergenic
1 rs766325 18956458 PAX7 intergenic
1 rs6695765 18979320 PAX7 intron
1 rs742071 18979874 PAX7 intron
1 rs560426 94553438 ABCA4 intron
1 rs481931 94570016 ABCA4 intron
1 rs4147811 94575056 ABCA4 intron
1 rs6677101 108699730 SLC25A24 intron
1 rs2235371 209964080 IRF6 extron
1 rs642961 209989270 IRF6 intergenic
1 rs126280 210019824 DIEXF intron
1 rs2064163 210048819 DIEXF intergenic
1 rs12063989 210049893 DIEXF intergenic
1 rs4844913 210068117 SYT14 intergenic
1 rs9429830 210110537 SYT14 intergenic
1 rs11119388 210174417 SYT14 intron
1 rs227178 210216946 SYT14 intron
1 rs2485893 210348155 SYT14 intergenic
2 rs7590268 43540125 THADA intron
3 rs7632427 89534377 EPHA3 intergenic
4 rs12506428 93830884 GRID2 intron
8 rs6558002 27389542 EPHX2 intron
8 rs12543318 88868340 DCAF4L2 intergenic
8 rs987525 129946154 LOC728724 intergenic
9 rs894673 100612270 FOXE1 intergenic
9 rs3758249 100614140 FOXE1 intergenic
9 rs4460498 100620412 FOXE1 intergenic
10 rs7078160 118827560 VAX1 intron
10 rs4752028 118834991 VAX1 intron
13 rs9574565 80668874 SPYR2 intergenic
13 rs8001641 80692811 SPYR2 intergenic
14 rs17563 54417522 BMP4 exon
15 rs1258763 33050423 FMN1 intergenic
15 rs7179658 63312695 TPM1 intergenic
16 rs8049367 3980445 CREBBP-ADCY9 intergenic
17 rs9788972 8919630 NTN1 intergenic
17 rs4791774 8932119 NTN1 intron
17 rs9915089 8952894 NTN1 intron
17 rs8069536 8956285 NTN1 intron
17 rs8081823 8965551 NTN1 intron
17 rs17760296 54615617 NOG intergenic
20 rs6072081 39261054 MAFB intergenic
20 rs6065259 39261979 MAFB intergenic
20 rs17820943 39268516 MAFB intergenic
20 rs13041247 39269074 MAFB intergenic
20 rs11698025 39274083 MAFB intergenic
20 rs6102085 39281629 MAFB intergenic
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Note: Chr, chromosome; SNP, single-nucleotide polymorphism.

Venous blood samples were collected from all participants after recruitment in the study. Genomic DNA was extracted using the protein precipitation method [20]. All the experiments of genotyping were done by the Genesky Biopharm Technology Company (http://www.geneskies.com/) with SNPscan technology.

Statistical analysis

The Hardy-Weinberg Equilibrium (HWE) analysis and the minor allele of frequency (MAF) determination were performed to check the deviation for each SNP among the unaffected parents. The HWE analysis was calculated through chi-square test. The MAF and allelic transmission disequilibrium test (TDT) and parent-of-origin effects analyses were calculated by PLINK software (http://zzz.bwh.harvard.edu/plink/data.shtml) [21]. The TDT analysis is a family-based association analysis method, which accessed whether the probability of two different alleles passed from heterozygous parents to their affected offspring varies by 50%. Pairwise linkage disequilibrium (LD) was calculated as D’ and r2 for 10 SNPs of 3 target genes (including NTN1, FOXE1 and VAX1) to identify LD blocks by the Haploview online software (http://www.broad.mit.edu/mpg/haploview/index.php). Linkage analysis is based on the principle of meiosis chromosomal exchange and recombination. To determine the correlation between genetic markers and the disease, the separation of genetic markers in a family was observed to see whether these markers were tightly linked with each other.

Results

Except for rs481931 and rs4147811, the genotypic distribution of remaining SNPs did not deviate from the Hardy-Weinberg equilibrium (P > 0.01) (Table 2).

Table 2.

Minor Allele Frequency and Hardy-Weinberg Equilibrium test for 47 SNPs

SNP A1 A2 MAF HWE
GENO O (HET) E (HET) P
rs4920522 T C 23.61% 14/76/137 0.33 0.35 0.45
rs766325 A G 21.13% 8/75/144 0.33 0.32 0.84
rs6695765 C T 37.17% 30/95/100 0.42 0.45 0.38
rs742071 T G 5.20% 0/16/211 0.070 0.068 1
rs560426 C T 32.85% 17/111/99 0.49 0.43 0.068
rs481931 T G 32.55% 11/115/99 0.51 0.42 0.0025
rs4147811 T C 33.19% 11/117/99 0.52 0.42 0.0016
rs6677101 T G 46.20% 51/111/64 0.49 0.50 0.89
rs2235371 T C 33.41% 31/97/99 0.43 0.46 0.38
rs642961 A G 24.23% 13/82/132 0.36 0.36 1
rs126280 A G 23.81% 11/79/134 0.35 0.35 1
rs2064163 T G 39.73% 37/107/82 0.47 0.48 0.89
rs12063989 C T 36.38% 35/100/91 0.44 0.47 0.40
rs4844913 G A 47.22% 46/106/74 0.47 0.49 0.50
rs9429830 T C 49.76% 63/87/58 0.42 0.50 0.019
rs11119388 G A 40.71% 44/99/84 0.44 0.48 0.13
rs227178 C T 42.98% 39/98/89 0.43 0.48 0.21
rs2485893 G A 47.35% 45/103/79 0.45 0.49 0.28
rs7590268 G T 3.44% 0/15/212 0.07 0.064 1
rs7632427 C T 16.48% 4/70/153 0.31 0.28 0.25
rs12506428 C T 49.45% 60/106/61 0.47 0.50 0.35
rs6558002 C T 16.19% 5/58/164 0.26 0.25 1
rs12543318 A C 34.40% 28/105/94 0.46 0.46 1
rs987525 A C 8.52% 2/28/197 0.12 0.13 0.30
rs894673 A T 12.72% 3/49/174 0.22 0.21 1
rs3758249 T C 13.08% 3/50/174 0.22 0.22 1
rs4460498 T C 12.83% 3/49/174 0.22 0.21 1
rs7078160 A G 49.11% 57/115/55 0.51 0.50 0.89
rs4752028 C T 38.94% 38/112/77 0.49 0.49 0.89
rs9574565 T C 12.72% 6/44/177 0.19 0.22 0.12
rs8001641 A G 14.67% 2/67/156 0.30 0.27 0.082
rs17563 G A 31.25% 23/94/109 0.42 0.43 0.76
rs1258763 T C 8.33% 2/38/186 0.17 0.17 1
rs7179658 C T 15.82% 5/60/162 0.26 0.26 1
rs8049367 T C 34.29% 35/96/96 0.42 0.46 0.20
rs9788972 A G 22.57% 14/68/145 0.30 0.33 0.16
rs4791774 G A 22.62% 11/73/143 0.32 0.33 0.69
rs9915089 T C 19.47% 8/67/152 0.30 0.30 0.83
rs8069536 T G 3.65% 0/13/214 0.057 0.06 1
rs8081823 A G 39.89% 31/112/83 0.50 0.47 0.57
rs17760296 G T 1.55% 0/8/219 0.035 0.035 1
rs6072081 G A 39.05% 31/116/80 0.51 0.48 0.33
rs6065259 A G 36.76% 29/107/86 0.48 0.47 0.67
rs17820943 T C 39.38% 31/116/80 0.51 0.48 0.33
rs13041247 C T 39.38% 31/116/80 0.51 0.48 0.33
rs11698025 A G 32.15% 21/99/107 0.44 0.43 0.88
rs6102085 A G 42.86% 42/115/69 0.51 0.49 0.69
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Note: SNP, single-nucleotide polymorphism; A1, minor allele; A2, major allele; MAF, minor allele frequency; HWE, Hardy-Weinberg Equilibrium test; GENO, genotype; O (HET), observed heterozygosity; E (HET), expect heterozygosity.

Allelic TDT analysis was usually assessed the transmission of minor alleles from heterozygous informative parents to affected child within case-parents trios. The results showed allele A at rs894673 of FOXE1 (P = 0.0071, ORtransmission = 0.35, 95% CI: 0.16-0.78), and allele T at rs3758249 of FOXE1 (P = 0.0071, ORtransmission = 0.35, 95% CI: 0.16-0.78) were under-transmitted. Allele A at rs7078160 and allele C at rs4752028 of VAX1 showed a tendency of over-transmission (P = 0.039, ORtransmission = 1.62, 95% CI: 1.02-2.58; P = 0.024, ORtransmission = 1.73, 95% CI: 1.07-2.81; respectively) (Table 3).

Table 3.

Allelic transmission disequilibrium test results for 47 SNPs

SNP A1 T:U CHISQ P OR (95% CI) Parental COM
A:U CHISQ P CHISQ P
rs4920522 T 26:23 0.18 0.67 1.13 (0.65-1.98) 69:60 0.46 0.50 0.64 0.42
rs766325 A 24:23 0.02 0.88 1.04 (0.59-1.85) 67:55 0.92 0.34 0.83 0.36
rs6695765 C 34:29 0.40 0.53 1.17 (0.71-1.92) 94:62 5.02 0.025 5.13 0.024
rs742071 T 10:5 1.67 0.20 2.00 (0.68-5.85) 31:16 4.41 0.036 6.06 0.014
rs560426 C 42:28 2.80 0.094 1.50 (0.93-2.42) 74:63 0.74 0.39 2.68 0.10
rs481931 T 31:39 0.91 0.34 0.79 (0.50-1.27) 80:60 2.27 0.13 0.59 0.44
rs4147811 T 35:41 0.47 0.49 0.85 (0.54-1.34) 83:58 3.57 0.059 1.44 0.23
rs6677101 T 42:30 2.00 0.16 1.40 (0.88-2.24) 74:81 0.24 0.63 0.090 0.77
rs2235371 T 39:38 0.013 0.91 1.03 (0.66-1.60) 70:81 0.62 0.43 0.37 0.54
rs642961 A 23:26 0.18 0.67 0.88 (0.50-1.55) 66:62 0.10 0.75 0.0049 0.94
rs126280 A 20:28 1.33 0.25 0.71 (0.40-1.27) 70:55 1.53 0.22 0.25 0.62
rs2064163 T 32:37 0.36 0.55 0.86 (0.54-1.39) 82:80 0.019 0.89 0.032 0.86
rs12063989 C 31:38 0.71 0.40 0.82 (0.51-1.31) 73:80 0.25 0.62 0.73 0.39
rs4844913 G 40:33 0.67 0.41 1.21 (0.76-1.92) 100:70 4.05 0.044 4.64 0.031
rs9429830 T 30:25 0.45 0.50 1.20 (0.71-2.04) 67:78 0.61 0.44 0.14 0.71
rs11119388 G 38:40 0.051 0.82 0.95 (0.61-1.48) 80:86 0.16 0.69 0.22 0.64
rs227178 C 36:32 0.24 0.63 1.13 (0.70-1.81) 105:67 6.28 0.012 5.92 0.015
rs2485893 G 39:32 0.69 0.41 1.22 (0.76-1.95) 108:62 9.36 0.0022 9.46 0.0021
rs7590268 G 1:8 5.44 0.020 0.13 (0.016-1.00) 14:12 0.15 0.70 0.71 0.40
rs7632427 C 14:18 0.50 0.48 0.78 (0.39-1.56) 46:58 1.16 0.28 1.64 0.20
rs12506428 C 32:41 1.11 0.29 0.78 (0.49-1.24) 78:83 0.11 0.74 0.66 0.42
rs6558002 C 10:20 3.33 0.068 0.50 (0.23-1.07) 50:42 0.60 0.44 0.029 0.86
rs12543318 A 30:37 0.73 0.39 0.81 (0.50-1.31) 70:77 0.24 0.62 0.73 0.39
rs987525 A 15:7 2.91 0.088 2.14 (0.87-5.26) 41:28 2.25 0.13 4.55 0.033
rs894673 A 8:23 7.26 0.0071 0.35 (0.16-0.78) 38:37 0.012 0.91 1.75 0.19
rs3758249 T 8:23 7.26 0.0071 0.35 (0.16-0.78) 39:38 0.012 0.91 1.72 0.19
rs4460498 T 8:22 6.53 0.011 0.36 (0.16-0.82) 37:37 0 1.00 1.78 0.18
rs7078160 A 47:29 4.26 0.039 1.62 (1.02-2.58) 69:77 0.35 0.56 0.38 0.54
rs4752028 C 45:26 5.09 0.024 1.73 (1.07-2.81) 67:82 1.19 0.28 0.062 0.80
rs9574565 T 16:14 0.13 0.72 1.14 (0.56-2.34) 47:42 0.25 0.62 0.37 0.54
rs8001641 A 18:14 0.50 0.48 1.29 (0.64-2.59) 43:53 0.93 0.34 0.26 0.61
rs17563 G 28:29 0.018 0.89 0.97 (0.57-1.62) 73:74 0.0056 0.94 0.017 0.90
rs1258763 T 8:8 0 1.00 1.00 (0.38-2.67) 29:38 1.08 0.30 0.89 0.35
rs7179658 C 23:17 0.90 0.34 1.35 (0.72-2.53) 57:54 0.074 0.79 0.50 0.48
rs8049367 T 33:24 1.42 0.23 1.38 (0.81-2.33) 69:89 1.96 0.16 0.46 0.50
rs9788972 A 19:30 2.47 0.12 0.63 (0.36-1.13) 72:60 0.91 0.34 0.0048 0.94
rs4791774 G 21:26 0.53 0.47 0.81 (0.45-1.44) 69:55 1.34 0.25 0.42 0.52
rs9915089 T 16:30 4.26 0.039 0.53 (0.29-0.98) 68:56 0.94 0.33 0.020 0.89
rs8069536 T 4:7 0.82 0.37 0.57 (0.17-1.95) 20:13 1.49 0.22 0.36 0.55
rs8081823 A 36:37 0.014 0.91 0.97 (0.62-1.54) 96:84 0.61 0.44 0.39 0.53
rs17760296 G 2:2 0 1.00 1.00 (0.14-7.10) 6:8 0.29 0.59 0.22 0.64
rs6072081 G 35:46 1.49 0.22 0.76 (0.49-1.18) 75:73 0.021 0.88 0.30 0.58
rs6065259 A 32:42 1.35 0.25 0.76 (0.48-1.21) 73:73 0 1.00 0.38 0.54
rs17820943 T 36:46 1.22 0.27 0.78 (0.51-1.21) 74:71 0.048 0.83 0.18 0.67
rs13041247 C 36:46 1.22 0.27 0.78 (0.51-1.21) 74:71 0.048 0.83 0.18 0.67
rs11698025 A 35:41 0.47 0.49 0.85 (0.54-1.34) 71:60 0.74 0.39 0.10 0.75
rs6102085 A 36:38 0.054 0.82 0.95 (0.60-1.49) 79:82 0.043 0.84 0.088 0.77
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Note: SNP, single-nucleotide polymorphism; A1, minor allele; T:U, transmitted:untransmitted; OR, odds ratio; A:U, discordance counts; COM, combined analysis results; Bold characters indicate the items with P-value less than 0.05.

In parent-of-origin effects analysis, allele A at rs894673, allele T at rs3758249 and allele T at rs4460498 of FOXE1 showed a weak paternal special under-transmission bias (P = 0.039; P = 0.039; P = 0.039; respectively). Allele A at rs987525 (downstream of LOC728724) showed a relatively strong paternal special over-transmission bias (P = 0.0077) (Table 4). However, no significant difference was found within combined analysis between father and mother.

Table 4.

Parent-of-Origin effects analysis for 47 SNPs

SNP A1:A2 Paternal Maternal Z P
T:U CHISQ P T:U CHISQ P
rs4920522 T:C 15:11 0.62 0.43 11:12 0.043 0.83 0.69 0.49
rs766325 A:G 12.5:12.5 0 1 11.5:10.5 0.045 0.83 -0.16 0.88
rs6695765 C:T 15.5:12.5 0.32 0.57 18.5:16.5 0.11 0.74 0.20 0.84
rs742071 T:G 2:3 0.20 0.65 8:2 3.60 0.058 -1.48 0.14
rs560426 C:T 17:14 0.29 0.59 25:14 3.10 0.078 -0.78 0.43
rs481931 T:G 17:26 1.88 0.17 14:13 0.037 0.85 -1.01 0.31
rs4147811 T:C 19.5:24.5 0.57 0.45 15.5:16.5 0.031 0.86 -0.36 0.72
rs6677101 T:G 20:18 0.11 0.75 22:12 2.94 0.086 -1.03 0.30
rs2235371 T:C 19:20 0.026 0.87 20:18 0.11 0.75 -0.34 0.73
rs642961 A:G 9.5:13.5 0.70 0.40 13.5:12.5 0.038 0.84 -0.74 0.46
rs126280 A:G 8:12 0.80 0.37 12:16 0.57 0.45 -0.20 0.84
rs2064163 T:G 15:21 1 0.32 17:16 0.030 0.86 -0.82 0.41
rs12063989 C:T 15.5:20.5 0.69 0.40 15.5:17.5 0.12 0.73 -0.33 0.74
rs4844913 G:A 21.5:14.5 1.36 0.24 18.5:18.5 0 1 0.83 0.40
rs9429830 T:C 13.5:13.5 0 1 16.5:11.5 0.89 0.34 -0.66 0.51
rs11119388 G:A 18.5:18.5 0 1 19.5:21.5 0.098 0.75 0.22 0.83
rs227178 C:T 19.5:14.5 0.74 0.39 16.5:17.5 0.029 0.86 0.73 0.47
rs2485893 G:A 22:13 2.31 0.13 17:19 0.11 0.74 1.32 0.19
rs7590268 G:T 1:4 1.80 0.18 0:4 4 0.046 NA NA
rs7632427 C:T 7:12 1.32 0.25 7:6 0.077 0.78 -0.95 0.34
rs12506428 C:T 14.5:21.5 1.36 0.24 17.5:19.5 0.11 0.74 -0.60 0.55
rs6558002 C:T 5:12 2.88 0.09 5:8 0.69 0.41 -0.52 0.60
rs12543318 A:C 17:19 0.11 0.74 13:18 0.81 0.37 0.43 0.66
rs987525 A:C 8.5:0.5 7.11 0.0077 6.5:6.5 0 1 1.82 0.069
rs894673 A:T 3.5:11.5 4.27 0.039 4.5:11.5 3.06 0.080 -0.30 0.76
rs3758249 T:C 3.5:11.5 4.27 0.039 4.5:11.5 3.06 0.080 -0.30 0.76
rs4460498 T:C 3.5:11.5 4.27 0.039 4.5:10.5 2.4 0.12 -0.41 0.68
rs7078160 A:G 24.5:13.5 3.18 0.074 22.5:15.5 1.29 0.26 0.47 0.64
rs4752028 C:T 18.5:13.5 0.78 0.38 26.5:12.5 5.03 0.025 -0.88 0.38
rs9574565 T:C 7:10 0.53 0.47 9:4 1.92 0.17 -1.50 0.13
rs8001641 A:G 11.5:5.5 2.12 0.15 6.5:8.5 0.27 0.61 1.37 0.17
rs17563 G:A 10.5:12.5 0.17 0.68 17.5:16.5 0.029 0.86 -0.43 0.67
rs1258763 T:C 3:4 0.14 0.71 5:4 0.11 0.74 -0.50 0.62
rs7179658 C:T 13.5:9.5 0.70 0.40 9.5:7.5 0.24 0.63 0.18 0.86
rs8049367 T:C 17.5:11.5 1.24 0.27 15.5:12.5 0.32 0.57 0.38 0.70
rs9788972 A:G 9:13 0.73 0.39 10:17 1.82 0.18 0.28 0.78
rs4791774 G:A 10:13 0.39 0.53 11:13 0.17 0.68 -0.16 0.87
rs9915089 T:C 7:16 3.52 0.061 9:14 1.09 0.30 -0.62 0.54
rs8069536 T:G 0:3 3 0.083 4:4 0 1 NA NA
rs8081823 A:G 19.5:23.5 0.37 0.54 16.5:13.5 0.30 0.58 -0.81 0.42
rs17760296 G:T 1:1 0 1 1:1 0 1 0 1
rs6072081 G:A 21.5:25.5 0.34 0.56 13.5:20.5 1.44 0.23 0.54 0.59
rs6065259 A:G 17:25 1.52 0.22 15:17 0.13 0.72 -0.55 0.58
rs17820943 T:C 20:24 0.36 0.55 16:22 0.95 0.33 0.30 0.76
rs13041247 C:T 20:24 0.36 0.55 16:22 0.95 0.33 0.30 0.76
rs11698025 A:G 16.5:22.5 0.92 0.34 18.5:18.5 0 1 -0.67 0.50
rs6102085 A:G 20.5:18.5 0.10 0.75 15.5:19.5 0.46 0.50 0.71 0.48
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Note: SNP, single-nucleotide polymorphism; A1, minor allele; A2, major allele; T:U, transmitted:undertransmitted; Z, vector of the large sample Z statistic; NA, not available; Bold characters indicate the items with P-value less than 0.05.

We calculated the pairwise LD of SNPs on 3 target genes (including NTN1, FOXE1 and VAX1) based the association results. Pairwise LD analysis indicated that the SNPs of FOXE1 (rs894673, rs3758249, and rs4460498) were significantly linked with each other (r2 > 0.95, D’ = 1) (Figure 1).

Figure 1.

Figure 1

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Pairwise LD analysis of the target genes (NTN1, FOXE1 and VAX1) for cleft alveolus (A, D’; B, r2).

Discussion

Phenotype is an observable or measurable trait of a disease. Some typical phenotype (e.g. lower lip pits for Van der Woude syndrome; bilateral zygomatic and malar hypoplasia for Treacher Collins syndrome) is important and usually will lead to a clinical diagnosis, which could provide information to infer the candidate genes with phenotypic similarities and consult the phenotype-genotype heterogeneous network [22,23]. Previous genetic studies of NSCL/P paid more attention to NSCLO, NSCPO and NSCLP, and even combined analysis with mixed samples. As a specific phenotype, cleft alveolus has not been studied by itself.

The maxillary processes fuse with the medial nasal process to form the upper lip, alveolus, and primary palate during the 5th and 6th weeks of embryonic development [1,24]. The process of forming alveolar bone has extensive similarity to the process in the upper lip, which arises from the same embryonic origin. Thus, we infer that cleft alveolus might share the same gene regulatory network with cleft lip.

FOXE1, located on 9q22, was initially suggested as a candidate gene through genome scanning and linkage analysis in some multiplex CL/P families [25]. Subsequently, significant SNPs that linked 9q22 region were replicated in CL/P families of Colombia, USA, and the Philippines. Rs3758249 and rs4460498 showed highly significant signals, which were located inside a 70 kb high linkage disequilibrium block containing FOXE1. Then, expression of foxe1 was detected in caudal epithelium of medial nasal and maxillary processes at E11.5. Especially, foxe1 is obviously expressed in the stage of fusion between the medial nasal and maxillary processes [26]. Animal experiments had demonstrated that FOXE1 in knockout mice will lead to developmental malformation, including thyroid dysgenesis and cleft palate [27]. Then, FOXE1 overexpression experiments in a mouse model also displayed a phenotype of cleft palate, and FOXE1 was highly expressed in the medial edge epithelium (MEE) [28]. This evidence suggested that FOXE1 might regulate the pathogenesis of cleft lip and palate.

Notably, the impact of FOXE1 on the NSCL/P should be validated in different ethnic groups in order to exclude the limitation of single populations’ or regions’ mutation. In a follow-up association study of fifty SNPs at 9q22, rs894673 and rs3758249 near FOXE1 were genotyped in 291 multiplex cleft families. However, no significant association of FOXE1 was found in the test populations [29]. Subsequently, the association of target SNPs (rs894673, rs3758249 and rs4460498) near FOXE1 with NSCL/P were validated among distinct populations [30-33]. These results supported that FOXE1 gene was a positive risk factor for orofacial cleft. In our study, rs894673 and rs3758249 showed a significant association with cleft alveolus, and rs4460498 showed a moderate association with cleft alveolus (Table 3). Meanwhile, marked parent of origin effects were seen with rs894673, rs3758249, and rs4460498 alleles. Under-transmission was shown preferentially from comparing fathers to mothers (Table 4). Pair-wise LD analysis also showed a strong LD between rs894673, rs3758249 and rs4460498 (Figure 1), indicating they were tightly linked with each other. All these findings suggested the pathogenesis of cleft alveolus involved FOXE1 gene expression and regulation.

Rs894673 and rs3758249 are located in the 5’-upstream region of FOXE1, speculating they might affect FOXE1 transcription by altering transcription factor binding sites. The entire promoter region of FOXE1 was screened with 35 cleft palate patients and 160 unaffected people to identify the suspicious variants A novel non-coding variant in the 5’-untranslated region of FOXE1 was found. Based on later cell experiments, the variant could prevent the binding of MYF-5 to FOXE1 promoter and affect the FOXE1 expression [34]. Rs4460498, located in the 3’-downstream region of FOXE1, was first reported to have an association with NSCL/P among Caucasian and Asian populations with P value 6.51E-12 [26]. Three potentially functional SNPs of FOXE1 (two in the 5’-upstream and one in 3’-UTR) were replicated among central Chinese population in a following study. They found that the target SNP in 3’-UTR contributed to altering binding ability with target miRNA in in vitro studies [35].

Numerous studies had reported a significant association between rs987525 and NSCL/P in more than one population origin [14,15,18]. In this study, we found rs987525 had a mild association with parents and cases in combined TDT analysis (Table 3). But the association did not present in the cases. Meanwhile, an allele of rs987525 showed a significant over-transmission from fathers compared to mothers (Table 4). Rs987525 located in 8q24, which had been demonstrated this region containing a remote Myc-regulated enhancer. Deletion of this region would lead to alternation of facial morphology, and even to CL/P phenotype [36]. Myc expression also can be regulated by such cis-enhancer element interacting with the Myc promoter by transcription factor Tcf-4 binding [37].

Rs481931 and rs4147811 deviated from the Hardy-Weinberg equilibrium (P < 0.01) in our samples, indicating it may need larger sample size to validate its significance. Previously, we had validated the association between these two SNPs and NSCL/P among 440 orofacial cleft trios. Unfortunately, rs481931 and rs4147811 were not compatible with the Hardy-Weinberg equilibrium as well [38]. Maybe there was a higher genetic load from parents to the probands in the case-parents design of the research.

In summary, we replicated 47 SNPs contributing to NSCL/P to investigate their roles in cleft alveolus in a western Han Chinese population. Based on the current study, we confirmed FOXE1 as a susceptible gene for the cleft alveolus, which provides a new research direction for the development of alveolar bone.

Acknowledgements

We are particularly thankful to patients and their families who participated in this study. This work was supported by the National Science Funds of China (No. 81600849) and Sichuan Province Science and Technology support program (2020YJ0211).

Disclosure of conflict of interest

None.

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