Interactions Between Small Ubiquitin-Like Modifier 1 and Nonsyndromic Orofacial Clefts

Abstract

Small ubiquitin-like modifier 1 (SUMO1) and environmental factors have been shown to be associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in several populations. This study aimed at confirming the contribution of SUMO1 gene and environmental factors to nonsyndromic orofacial clefts risk in western Han Chinese. Four single-nucleotide polymorphisms were investigated in 212 case trios in western China using conditional logistic regression models and the transmission disequilibrium test under a case–parent trio design. Strong evidence of linkage and linkage disequilibrium was found between these markers and the disease in both single-nucleotide polymorphism analysis (T allele at rs6761234 [p = 0.0005, odds ratio [OR] = 1.82, 95% confidence interval [CI]:1.30–2.57) and C allele at rs12470401 (p < 0.0001, OR = 2.82, 95% CI: 1.90–4.19)] and sliding window haplotype analysis (T-T-T for rs6761234-rs12470401-rs7599810 [p = 0.018], C-C-G for rs12470401-rs7599810-rs6435133 [0.0033], C-T-T-T for rs6761234-rs12470401-rs7599810-rs6435133 [p = 0.018] among others). Interactions between mothers' passive smoking during the first trimester and C/C genotype of rs12470401 showed statistical significance (OR₀ = 2.53 and OR₁ = 8.83). Risk factors identified in this study may provide a better understanding of the etiological role of SUMO1 gene in NSCL/P incidence.

Introduction

N onsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects with a prevalence of 1/500–1/1400 live births and varies with ethnicity (Gorlin et al., 2001; Wyszynski, 2002; Hashmi et al., 2005). Its etiology is considered to be multifactorial, resulting from both genetics and environmental exposures, neither of which has been fully identified. Candidate gene studies or genome-wide mapping searches for novel loci or regions have led to identification of several genetics factors involved in clefting, such as 1q32, 2p13, 2q32-33, 3q27-28, 6q21-24, 9q21, 12p11, 14q21-24, 16q21-24, and 22q12.2-q12.3 (Brewer et al., 1998, 1999; Prescott et al., 2000; Field et al., 2002, 2004; Wyszynski et al., 2003; Marazita et al., 2004a, 2004b, 2009; Van Buggenhou et al., 2005; Radhakrishna et al., 2006; Riley et al., 2007; Brett et al., 2008).

Small ubiquitin-like modifier 1 (SUMO1) gene, which is located on 2q33, encodes a protein that is a member of the SUMO protein family. It functions in a manner similar to ubiquitin in that it is bound to target proteins as part of a post-translational modification system (NCBI).

We considered SUMO1 a potential candidate (gene), because it was abundantly expressed on mouse embryonic day 13.5 in the upper lip, primary palate, and medial edge epithelia of the secondary palate. When the SUMO1 gene is disrupted by balanced translocation, it may lead to cleft lip and palate (Alkuraya et al., 2006). Moreover, case–control and family-based association studies have identified linkage disequililibrum (LD) between polymorphic alleles at the SUMO1 locus and NSCL/P (Song et al., 2008; Carter et al., 2010).

All these reports suggest that SUMO1 may predispose to CL/P. The risk of CL/P associated with particular variants in SUMO1 may differ among different ethnic groups, and it is rarely reported to be associated with NSCL/P in the western Han Chinese.

It is known that NSCL/P is a complex and heterogeneous congenital malformation; its occurrence is highly associated with several environmental and genetic risk factors. Many epidemiological investigations and animal experiments have confirmed the exposure to environmental risk factors during early pregnancy, such as smoking, drinking, lack of vitamins, viral infection, and mothers' illness and medication during the first trimester can increase the risk of NSCL/P (Little et al., 2004; Shaw et al., 2005; Nandor et al., 2006; Grewal et al., 2008; Jia et al., 2009). In this study, transmission disequilibrium test (TDT) and conditional logistic regression models were used in case–parent trio design to find the relationship among environmental exposure factors during the first trimester and four single-nucleotide polymorphisms (SNPs) in SUMO1 gene in 212 case–parent trios from western China.

Materials and Methods

Subjects

Our sample consists of 212 complete case trios, all of whom were recruited between 2008 and 2009 from the Cleft Surgery Department of West China College of Stomatology, Sichuan University. Informed consent was obtained from each participant before enrollment in the study, as approved by the local ethics committee. To assess nonsyndromic status of cases, all probands were screened for the presence of associated anomalies or syndromes by a physician, and only those determined to have an isolated CL/P were included in this study. Patients were asked about family history of oral clefts among first and second relatives. All subjects were self-identified as western Han Chinese. Table 1 shows gender and family history among NSCL/P cases.

Table 1.

Gender and Family History Among Cases of Nonsyndromic Orofacial Clefts

NSCL/P	212
Sporadic case	194
Family history positive	18
Male case	146
Female case	66

NSCL/P, nonsyndromic cleft lip with or without cleft palate.

Data collection

A brief interview of mothers and fathers was conducted at their initial visit, including family history, fathers' drinking and smoking history, mothers' abortion history, and environmental exposures during the first trimester: medication history (if they took any medication against viral infection), illness history, vitamins (including folic acid), supplementation history, and smoking history. Mothers were asked whether they or their partner smoked at the beginning of pregnancy, whether they currently smoked, and about daily and/or weekly cigarette consumption. A separate question was asked about the total number of cigarettes smoked indoors. Smoking was coded as a dichotomous variable: If they smoked more than one cigarette within a week, they were coded as smokers; if the answer was “no,” they were coded as nonsmokers; and if the answers to all the questions on smoking were missing, they were coded as missing.

Genotyping

Venous blood samples were drawn from participants at their initial visit. DNA was extracted by phenol chloroform extraction protocol and genotyped for SNP markers using the restriction fragment length polymorphism–polymerase chain reaction (Supplementary Table S1; Supplementary Data are available online at www.liebertonline.com/dna). The accuracy of the method was confirmed by direct sequencing of amplified DNA from randomly selected samples (10%), and no difference in results was found between the two methods (data not shown).

Statistical analysis

Hardy–Weinberg equilibrium at each SNP was computed among parents. The TDT were performed by the family-based association test (FBAT) program (www.biostat.harvard.edu/∼fbat/default.html) and SAS 8.1 statistical software Package (SAS Institute, Inc., Cary, NC). Pairwise LD as well as their D′ and r ² were computed for all SNPs using the haploview program (www.broad.mit.edu/haploview/haploview) in the case–parent trios. Individual SNPs and sliding window haplotypes consisting of two, three, and four SNPs were tested using FBAT program. Genotypic odds ratios (ORs) for genotypes were obtained from conditional logistic regression models for matched sets consisting of the case and three “pseudosib” controls derived from the parental mating type using SPSS 11.5 statistical software Package. Interactions between the SUMO1 gene and the environmental risk factors were performed by SAS 8.1 statistical software Package.

Results

Hardy–Weinberg equilibrium and LD

Hardy–Weinberg equilibrium was assessed for each of the four SNPs among the parents of cases. Significant deviation from Hardy–Weinberg expectations could reflect genotyping errors or true heterogeneity in the population and could bias our statistical tests. All χ ² tests using the genotype frequency of parents showed that the four SNPs included in the study were in Hardy–Weinberg equilibrium. LD between markers was calculated by D′ and r ² statistics from haploview analysis. Among the four SNPs, one group of markers (rs6761234-rs7599810) showed tight LD (D′ = 0.74); whereas the other groups of markers showed relatively weak LD (D′ < 0.8 and r ² < 0.8) (Supplementary Table S2).

TDT analyses of individual markers and haplotypes

TDT results of the 2 × 2 χ ² test carried out on heterozygous case–parent trios showed that the T allele at rs6761234 (p = 0.0005, OR = 1.82, 95% confidence interval (CI): 1.30–2.57) and C allele at rs12470401 (p < 0.0001, OR = 2.82, 95% CI: 1.90–4.19) were overtransmitted from parents to affected progeny. This suggests that these alleles may significantly increase the risk of being NSCL/P cases (Table 2).

Table 2.

The Transmission Disequilibrium Test Results From 2 × 2 Table χ ² Test

SNP ID	Allele	T/NT
rs7599810	T	142/121
	C	121/142
	p	0.067
	OR	1.38
	95% CI	0.98—1.94
rs6761234	T	154/114
	C	114/154
	p	0.0005 ^a
	OR	1.82
	95% CI	1.30—2.57
rs6435133	T	149/127
	G	127/149
	p	0.061
	OR	1.38
	95% CI	0.98—1.92
rs12470401	C	131/78
	T	78/131
	p	<0.0001 ^b
	OR	2.82
	95% CI	1.90—4.19

95% CI: 95% confidence interval; OR, odds ratio.

<0.01.

<0.0001.

SNP, single-nucleotide polymorphism; T/NT, transmitted or nontransmitted counts from heterozygous parents.

Bold numbers are statistically significant.

Results of the conditional logistic regress analysis showed that the T/T genotype at rs6435133 may decrease the risk of being NSCL/P cases (p = 0.043, OR = 0.055, 95% CI: 0.022–0.68) (Table 3).

Table 3.

Estimated Genotypic Risks for Individual Nucleotide Polymorphisms in Small Ubiquitin-Like Modifier 1 in Nonsyndromic Orofacial Clefts

SNP ID	Genotype	cases	Control ^a	p	OR	95% CI
rs7599810	TT	38	142	0.24	29.36	0.15–82.60
	TC	87	217	0.95	0.80	0.01–75.94
	CC	27	97	0.42	0.043	0.00–86.00
rs6761234	TT	50	160	0.50	19.75	0.004–85.87
	TC	87	225	0.97	0.84	0.00–24.51
	CC	19	83	0.66	0.076	0.00–63.24
rs6435133	GG	35	79	0.21	9.24	0.29–29.28
	GT	68	240	0.66	2.16	0.67–69.86
	TT	51	143	0.043 ^b	0.055	0.022–0.68
rs12470401	TT	20	67	0.72	0.21	0.00–96.60
	TC	48	206	0.80	0.36	0.36–103.77
	CC	59	108	0.32	21.71	0.47–93.37

Pseudo control.

<0.05.

Bold numbers are statistically significant.

FBAT analysis showed that the C allele (p = 0.0038), C/C genotype (p = 0.00039) at rs12470401, and G/G genotype at rs6435133 (p = 0.023) were over-represented in the affected progeny. FBAT analysis also showed that the T/C genotype at rs12470401 was under-represented among affected progeny, which suggests that this genotype may decrease the risk of being affected (Table 4).

Table 4.

Marker Information and Transmission Disequilibrium Test Results for Single-Nucleotide Polymorphisms in Small Ubiquitin-Like Modifier 1-Mode In Bi-Allelic Genetic Model (Additive and Genotype)

SNP ID	Allele	Allele frequency	Informative family #	S	E(S)	Var(S)	Z	p
Additive
rs7599810	T	0.55	152	163	166	50	−0.42	0.67
	C	0.45	152	141	138	50	0.42	0.67
rs6761234	T	0.639	156	187	183	51.5	0.56	0.58
	C	0.361	156	125	129	51.5	−0.56	0.58
rs6435133	G	0.368	144	137	123.5	50.25	1.90	0.057
	T	0.632	144	151	164.5	50.25	−1.90	0.057
rs12470401	T	0.295	127	88	107	43	−2.90	0.0038 ^a
	C	0.705	127	166	147	43	2.90	0.0038 ^a
Genotype
rs7599810	TT	0.314	114	38	45	25.5	−1.39	0.17
	TC	0.472	152	87	76	38	1.78	0.074
	CC	0.215	86	27	31	18.5	−0.93	0.35
rs6761234	TT	0.396	130	50	52.5	29.375	−0.46	0.64
	TC	0.486	156	87	78	39	1.44	0.15
	CC	0.118	76	19	25.5	15.875	−1.63	0.10
rs6435133	GG	0.13	80	35	25.75	16.438	2.28	0.023
	GT	0.476	144	67	72	36	−0.83	0.40
	TT	0.393	121	42	46.25	26.688	−0.82	0.41
rs12470401	TT	0.092	66	20	21.75	13.688	−0.47	0.64
	TC	0.406	127	48	63.5	31.75	−2.75	0.0059 ^b
	CC	0.502	106	59	41.75	23.688	3.54	0.00039 ^a

0.005.

<0.01.

S, score; E(S), expected score; Var (S), variance (score).

Bold numbers are statistically significant.

In Table 5, haplotypes consisting of two SNPs (rs6761234-rs12470401 and rs7599810-rs6435133) yielded statistical significance among NSCL/P cases (p = 0.03 and 0.031); haplotypes consisting of three SNPs (rs6761234-rs12470401-rs7599810 and rs12470401-rs7599810-rs6435133) also yielded statistical significance among NSCL/P cases (p = 0.018 and 0.0033); and haplotypes consisting of four SNPs yielded statistical significance among NSCL/P cases (p = 0.018). Interestingly then, two-SNP haplotypes (e.g., T-C for rs6761234-rs12470401 or C-G composed of rs7599810 and rs6435133) were more informative than three- or four-SNP haplotypes. Haplotype analysis showed that some haplotypes, for example, T-C (for rs6761234-rs12470401), C-G (for rs7599810-rs6435133) were far more common, and these haplotypes had a stronger impact on risk to NSCL/P.

Table 5.

Markers and Haplotypes Showing Significant Evidence of Linkage and Linkage Disequilibrium in 212 Chinese Case–Parent Trios

Marker/haplotype
rs6761234	rs12470401	rs7599810	rs6435133	Haplotype frequency	Trios(N) ^a	p^b
Overtransmitted
T	C	—	—	0.46	159	0.030
—	—	C	G	0.18	98.2	0.031
T	T	T	—	0.092	52.7	0.018
—	C	C	G	0.11	80.7	0.0033
C	T	T	T	0.05	42.2	0.018
Undertransmitted
T	T	—	—	0.18	96	0.034
—	T	T	—	0.14	67.6	0.0033
—	T	T	T	0.081	45.9	0.015
—	T	C	G	0.057	31.7	0.022

Number of informative case–parent trios.

The p-value for a statistical model with whole haplotypes consisted of two, three, or four SNPs.

Interactions between SUMO1 polymorphism and environmental factors

Jia et al. (2010) used single-factor χ ² analysis to test the association between nonsyndromic orofacial clefts and environmental factors. Their results showed that mothers'smoking, passive smoking, drinking, illness, medication, abortion history, and vitamins (including folic acid) supplementation during the first trimester were significantly associated with nonsyndromic orofacial clefts in western Han Chinese.

In this study, the previous “significant” environmental factors (mothers'smoking, passive smoking, drinking, illness, medication, abortion history, vitamins [including folic acid] supplementation during the first trimester), and genotypic risk factors (C/C genotype at rs12470401) were used to test for interactions between markers in SUMO1 and environmental factors from case–parental trio designs. The interaction between infant's C/C genotype at rs12470401 and mothers' drinking during the first trimester was significant (OR₀ = 2.53 and OR₁ = 8.83) (Table 6).

Table 6.

Interaction Between Single-Nucleotide Polymorphisms and Environmental Factors from Case–Parental Trio Designs

	No exposure			Exposure
Environmental factors	Case			Case
Mothers' smoking	Control^a	0	1	Control^a	0	1
	0	4	12	0	3	8
	1	24	114	1	9	38
	p	0.46		p	0.55
	OR₀	1.58		OR₁	1.58
	95% CI	0.47–5.33		95% CI	0.35–7.19
Mothers' drinking	Control^a	0	1	Control^a	0	1
	0	11	17	0	5	3
	1	23	90	1	10	53
	p	0.036		p	0.0023
	OR₀	2.53		OR₁	8.83
	95% CI	1.04–6.14		95% CI	1.81–43.00
Mothers' passive smoking during the first trimester	Control^a	0	1	Control^a	0	1
	0	4	8	0	3	12
	1	15	72	1	18	80
	p	0.18		p	0.88
	OR₀	2.40		OR₁	1.11
	95% CI	0.64–9.01	95% CI	0.28–4.35
Mothers' abortion history	Control^a	0	1	Control^a	0	1
	0	6	14	0	1	6
	1	26	113	1	7	39
	p	0.24		p	0.95
	OR₀	1.86		OR₁	0.93
	95% CI	0.65–5.31		95% CI	0.096–8.94
Mothers' illness during the first trimester	Control^a	0	1	Control^a	0	1
	0	2	10	0	5	10
	1	11	73	1	22	79
	p	0.74		p	0.32
	OR₀	1.33		OR₁	1.80
	95% CI	0.26–6.88		95% CI	0.56–5.80
Mothers' medication during the first trimester	Control^a	0	1	Control^a	0	1
	0	2	11	0	9	5
	1	16	83	1	17	69
	p	0.94		p	0.18
	OR₀	0.94		OR₁	2.25
	95% CI	0.19–4.67		95% CI	0.67–7.60
Mothers' vitamins supplementation during the first trimester	Control^a	0	1	Control^a	0	1
	0	6	16	0	1	4
	1	24	95	1	9	57
	p	0.45		p	0.69
	OR₀	1.48		OR₁	1.58
	95% CI	0.52–4.20		95% CI	0.16–15.81

Case–parental control.

0, T/T genotype of rs12470401; 1, C/C and T/C genotype of rs12470401.

Discussion

NSCL/P is a complex and heterogenerous trait with no obvious mode of inheritance. Numerous studies have failed to identify genes with any major influence on risk to NSCL/P (Jugessur and Murray et al., 2005). The SUMO1 gene was analyzed in the present study, because it was expressed on mouse embryonic day 13.5 in the upper lip, primary palate, and medial edge epithelia of the secondary palate; additional studies on animal models confirmed the role of SUMO1 in palate formation (Alkuraya et al., 2006).

The four SNPs examined here are located in the intron of the SUMO1 gene. Although intronic SNPs do not typically alter protein structure, associations with intronic variants have been reported for a number of complex diseases. Among these four SNPs in our entire sample, C allele and C/C genotype at rs12470401, T allele at rs6761234, and G/G genotype at rs6435133 were overtransmitted from parents to affected progeny with a significant increase being NSCL/P cases.

In the studied sample of case–parent trios, haplotypes carrying the G allele at rs6435133 and C allele at rs12470401 were found to be overtransmitted from parents to affected progeny, whereas those haplotypes carrying the T allele at rs12470401 were negatively associated. Here, the alleles were reported on the forward strand of the chromosome (NCBI build 36.3), although the gene is transcribed from the reverse strand. Allele designations need to be reversed when compared with published reports that used the transcription strand as the reference.

Many studies identified a relationship between environmental factors and NSCL/P (Little et al., 2004; Johnson and Little, 2008; Jia et al., 2009, 2010). In this study, statistically significant evidence was found from case–parental trio design for the gene- environment interaction between C/C genotype at rs12470401 and mothers' drinking during the first trimester (OR₀ = 2.53 and OR₁ = 8.83) (Table 6), which provided new estimate of the possible impact of environmental factors and SUMO1 polymorphism on oral clefts. This argues that genes such as SUMO1 may become more critical in the etiology of NSCL/P in the presence of certain maternal exposures.

Footnotes

Acknowledgments

This research was supported by the Key Program of National Natural Science Foundation of China (Grant No. 30530730), Scientific Research Funds for young teachers of Sichuan University (No. 2008067), and Sichuan University (No. 2009SCU11159).

Disclosure Statement

No competing financial interests exist.

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