Case–Control and Family-Based Association Studies of Novel Susceptibility Locus 8q24 in Nonsyndromic Cleft Lip With or Without Cleft Palate in a Southern Han Chinese Population Located in Guangdong Province

Abstract

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital malformations and a susceptibility locus on chromosome 8q24 has been replicated as a genetic risk factor for NSCL/P in patients of European and Asian descent. However, given considerable variations in allele frequencies across geographical regions studied, the aim of this study was to investigate the association of rs987525 located at 8q24 with NSCL/P only among the southern Han Chinese population from Guangdong province. We recruited 216 NSCL/P cases, their parents, and 200 controls to conduct case–control analysis and family-based association studies. Genotyping of rs987525 was carried out by the matrix assisted laser desorption ionization-time of flight mass spectrometry method. Case–control analysis showed allele and genotype distributions for rs987525 were not significantly associated with the risk of NSCL/P in our study population. Similar results were found when all cases were stratified into cleft lip only and cleft lip with cleft palate. A transmission disequilibrium test showed no statistically significant transmission of A nor C alleles and family-based association test (FBAT) analysis provided no evidence of NSCL/P risk with single markers. These results do not provide evidence for an association between rs987525 at 8q24 and the risk of NSCL/P in the southern Han Chinese population from Guangdong province.

Introduction

N onsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital malformations worldwide. The prevalence of NSCL/P is estimated to vary between 1/500 and 1/2000 depending on ethnic factors, geographic origin, and socioeconomic level (Harville et al., 2007). Populations with Asian and Native American ancestry have the highest rates whereas populations from Africa show the lowest. In the Chinese population, the prevalence of NSCL/P is ∼1.423/1000 births (Dai et al., 2010).

There is considerable international variation in the clinical treatment available to children born with oral clefts. These children require multidisciplinary care from birth but this can extend into adulthood, which inevitably poses long-term impacts on the individual, their family, and on society, with substantial expenditure in terms of health and social services (Jugessur et al., 2009; Mossey et al., 2009). The etiology of NSCL/P is complex and is currently considered to involve a strong genetic background risk and environmental factors (Mossey et al., 2009).

To date, a number of NSCL/P-associated genes and chromosomal regions such as PVRL1 (Sozen et al., 2001), TGF-β₃ (Vieira et al., 2003), MSX1 (van den Boogaard et al., 2000), TBX22 (Braybrook et al., 2001), FGFs (Riley and Murray, 2007), PDGF-C (Choi et al., 2009), 8q24 (Birnbaum et al., 2009), 10q25.3 (Mangold et al., 2010), 17q22 (Mangold et al., 2010), MAFB (Beaty et al., 2010), ABCA4 (Beaty et al., 2010), and interferon regulatory factor 6 (IRF6) (Zucchero et al., 2004) have been reported. However, only the IRF6 gene has demonstrated consistent association with the risk of NSCL/P across studies (Zucchero et al., 2004; Rahimov et al., 2008).

Recently, the single-nucleotide polymorphism (SNP) rs987525 showed a significant signal for NSCL/P at a novel susceptibility locus on 8q24.21 which was reported in genome-wide association scan (GWAS) studies conducted in two independent populations of European origin (p=3.34×10⁻²⁴, odds ratio [OR]_het=2.57 and OR_hom=6.05 for a population of German origin (Birnbaum et al., 2009) and p=9.18×10⁻⁸, OR=2.09 for a population of American European descent (Grant et al., 2009)]. The association between rs987525 and NSCL/P was also replicated in Estonian, Lithuanian, Polish, non-Hispanic whites, Mesoamerican, and Asian populations including Han Chinese from western China, Shandong, and Wuhan (Nikopensius et al., 2009; Mostowska et al., 2010; Beaty et al., 2010; Blanton et al., 2010; Rojas-Martinez et al., 2010). However, no study has yet determined whether this locus confers a similar effect in the southern Han Chinese population located in Guangdong province. Since genetic investigations of the Han Chinese population revealed by genome-wide autosomal SNPs have shown that the southern Han Chinese in Guangdong province are genetically separate from Han Chinese in other provinces including western China, Shandong, and Wuhan (Chen et al., 2009), it was necessary to replicate the association of rs987525 with the risk of NSCL/P in the local population of Guangdong province.

Materials and Methods

Study participants

The participants of this study consisted of 216 children with sporadic NSCL/P (cases), their parents (193 fathers and 192 mothers, including 178 complete case-parent trios), and 200 controls. All recruited cases were southern Han Chinese born in Guangdong province and whose parents were also born in Guangdong province and identified themselves as local southern Han Chinese. Cases were recruited between March and October 2009 from the Cleft Lip and Palate Treatment Centre in the Second Affiliated Hospital of Shantou University Medical College, located in the eastern part of Guangdong province. Physicians screened all patients presenting at the Treatment Centre during the study period for the presence of associated anomalies or syndromes and recruited only those with isolated oral cleft for enrolment into the study. The severity of the cleft was classified according to a modified right lip, palate, left lip coding system proposed by Sivertsen et al. (2008). We did not include children with isolated cleft palate in our case group because the developmental mechanisms and familial distributions for cleft palate alone are distinct from cleft lip with or without cleft palate.

Controls were healthy southern Han Chinese born in Guangdong province with no known history of oral clefts in their family members, recruited from staff and students of Shantou University Medical College. A 10 mL blood sample was drawn in an ethylenediamine tetraacetic acid collection tube from each participant (cases, controls, and cases' parents) and DNA was subsequently extracted for genotyping as previously described (Xu et al., 2004).

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Shantou University Medical College and written informed consent was obtained from each person before enrolment in the study.

Genotyping

SNP rs987525 was investigated in this study. Analysis was performed by Shanghai Benegene Biotechnology Co., Ltd. using Mass ARRAY system (Sequenom) by means of matrix assisted laser desorption ionization-time of flight mass spectrometry method according to the manufacturer's instructions.

To validate genotyping results a random sample of ∼10% of the total study population, including case and control individuals, was selected and repeat analyses were performed by direct sequencing. Cycle sequencing was carried out according to the manufacturer's instructions using ABI Prism™ BigDye™ Terminator Cycle Sequencing kit and ABI Prism 3730 capillary sequencer (Applied Biosystems, Foster City, CA). Samples that failed the genotyping assay were excluded from statistical analyses.

Statistical analysis

The Hardy–Weinberg equilibrium (HWE) was assessed in parents of affected cases and in the control group. Genotype and allele frequencies among all cases (NSCL/P) and the clinical subgroups of cleft lip only (CL) and cleft lip with cleft palate (CLP) were each compared with those frequencies in the control group using χ² tests. The genotypic OR with 95% confidence intervals (95% CI) for cases compared with controls were also calculated for genotype frequencies.

A transmission disequilibrium test (TDT) was used to identify excess transmission of the target alleles from heterozyous parents to the affected offspring using only complete case-parent trios in analyses. To use data from all genetic information provided by cases, including those with missing parents, we also conducted FBAT analysis to test the null hypothesis that transmission of alleles from all parents to child was independent of phenotype (Spielman and Ewens, 1996).

We considered p-values <0.05 to be statistically significant. Statistical power was calculated using the computer program QUANTO 1.2.4 (http://hydra.usc.edu/gxe) (Gauderman, 2003) using a log additive model or a dominant model. Case–control statistical analyses were performed with the SPSS 11.0 statistical software package (SPSS, Inc., Chicago, IL) and TDT was calculated directly using a McNemar's test. FBAT was carried out using FBAT 2.0.2 software (www.biostat.harvard.edu/fbat/default.html).

Results

The baseline characteristics of the 216 NCL/P cases and 200 controls in addition to the separate CL and CLP case subgroups are shown in Table 1. The observed genotypic and allelic distributions of rs987525 in cases and controls are shown in Table 2. The genotypic frequencies in controls were all in HWE (p=0.857), suggesting good homogeneity within the study subjects.

Table 1.

Characteristics of the Individual NSCL/P Cases and Controls

	CL/P	CL	CLP	Controls
No. of probands	216	44	172	200
Age, mean years (SD)	7.95 (0.6)	7.7 (0.9)	8.2 (0.5)	18.5 (2.1)
Sex
Male	147 (68.1%)	26 (59.1%)	121 (70.3%)	130 (65%)
Female	69 (31.9%)	18 (40.9%)	51 (29.7%)	70 (35%)
Laterality
Right-side cleft	55 (25.5%)	12 (27.3%)	43 (25.0%)
Left-side cleft	105 (48.6%)	28 (63.6%)	77 (44.8%)
Bilateral cleft	56 (25.9%)	4 (9.1%)	52 (30.2%)
Severity
Mild	24 (11.1%)	24 (54.5%)	0
Moderate	26 (12.0%)	20 (45.5%)	6 (3.5.%)
Severe	166 (76.9%)	0	166 (96.5%)

CL, cleft lip only; CLP, cleft lip with palate; CL/P, cleft lip with or without palate.

Table 2.

Genotypic and Allelic Distributions of rs987525 in Affected Individuals and Controls

	CL/P	CL	CLP	Controls
	n=216	n=44	n=172	n=200
Genotypes
CC	188 (87.0%)	37 (84.1%)	151 (87.8%)	175 (87.5%)
CA	26 (12.0%)	6 (13.6%)	20 (11.6%)	24 (12.0%)
AA	2 (0.9%)	1 (2.3%)	1 (0.6%)	1 (0.5%)
χ ² (p, df=2)	0.264 (0.876)	1.510 (0.470)	0.023 (0.989)
Odds ratio (95% CI) for comparison
CA vs. CC	1.008 (0.558–1.822)	1.182 (0.452–3.095)	0.966 (0.513–1.817)
AA vs. CC	1.862 (0.167–20.713)	4.730 (0.289–77.345)	1.159 (0.072–18.687)
CA+AA vs. CC	1.043 (0.585–1.857)	1.324 (0.533–3.290)	0.974 (0.524–1.809)
Alleles
C	402 (93.1%)	80 (90.9%)	322 (93.6%)	374 (93.5%)
A	30 (6.9%)	8 (9.1%)	22 (6.4%)	26 (6.5%)
χ ² (p, df=1)	0.065 (0.798)	0.747 (0.387)	0.003 (0.954)

95% CI, 95% confidence interval.

As shown in Table 2, the genotypes CC, CA, and AA in controls were 87.5%, 12.0%, and 0.5% respectively. The distributions of genotypes in all NSCL/P cases were similar to those in the controls: CC, CA, and AA were 87.0%, 12.0%, and 0.9%, respectively (p=0.876). The prevalence of the potential risk allele A in rs987525 among all cleft cases was 6.9%, whereas 6.5% of the controls had the A allele. This difference was not statistically significant (p=0.798). Odds ratios in Table 2 estimating the risk associated with each genotype show that neither the heterozygote (CA genotype) nor the mutated homozygote (AA genotype) was associated with increased odds of NSCL/P, compared with the corresponding wildtype homozygote (CC genotype). When we combined the heterozygote and mutated homozygote assuming a dominant model (CA+AA), we also observed a nonsignificant association between the variant genotype and NSCL/P (Table 2).

In the stratified analyses of CL and CLP, no statistically significant differences in genotype nor in allele distributions were observed between either subgroup of cases and the controls.

TDT analysis carried out on 55 heterozygous case–parent trios (Table 3) showed no evidence of overtransmission nor undertransmission of A or C alleles at rs987525 for NSCL/P cases. Our FBAT analysis using all genetic information from cases and their parents (178 complete case-parent triads, 20 cases with one parent, and 8 cases who were orphans) again showed no associations between rs987525 and all affected cases (Table 4), further confirming the allelic TDT and case–control results.

Table 3.

Transmission Disequilibrium Test of rs987525 (165 Individuals from 55 Heterozygous NCL/P Case–Parent Trios)

	No. of alleles transmitted
	A	C	Total	χ² ^a	p-Value
Observed	22	33	55	2.2	0.14
Expected	27.5	27.5

χ ² was analyzed using McNemar's test.

Table 4.

Family-Based Association Test for rs987525 in 208 Families Including 601 Individuals

Allele/genotype	Afreq	fam#	S	E(S)	Var(S)	Z	p-Value
A	0.067	45	22	24	12.5	−1.5	0.2
C	0.933	45	70	64	12.5	1.5	0.2
AA	0.004	2^a
AC	0.124	45	18	22.5	11.2	−1.2	0.2
CC	0.871	45	28	21.5	10.8	1.2	0.2

Excluded due to a minimum informative family number of 10 for FBAT software analysis.

afreq, allele frequency; fam#, informative family number; S, standard deviation; E(s), expected value; Var(S), variance; Z, a vector of the Family-Based Association Test statistic.

Discussion

In the present study of the southern Han Chinese located in Guangdong province rs987525 located at 8q24 was not associated with NSCL/P. To our best knowledge, this is the first comprehensive genetic association study examining the relationship between the SNP located at the 8q24 locus and NSCL/P risk in this specific ethnic population from Guangdong, using both case–control and family-based association study methods.

We successfully genotyped rs987525 located in 8q24. Our case–control association study showed that none of the different genotypes nor alleles were associated with the risk of NSCL/P. Further, the TDT study showed that the transmission of the A and C alleles in rs987525 was not associated with NSCL/P (p>0.05) in heterogeneous case-parent trios and the single marker FBAT analysis provided no evidence of statistically significant associations with NSCL/P of rs987525. Overall, the above consistency among results suggests that rs987525 was not associated with NSCL/P in this southern Chinese Han population located in Guangdong Province. This is inconsistent with results reported by Beaty et al. (2010) using a pooled Asian population that included Han Chinese from western China, Shandong, and Wuhan. The lack of association between rs987525 and NSCL/P in Guandong Province might be due to greater genetic heterogeneity among the Guangdong Han Chinese. Principal component analysis on genome-wide autosomal SNPs data from the 10 provinces of China including the southern Han Chinese located in Guangdong showed a north-south genetic differentiation. Further, the Guangdong Han Chinese were shown to be distinct from Han Chinese samples from the other provinces (Chen et al., 2009). The distinct genetic background of the Han Chinese born and living in Guangdong province is probably due to genetic exchange between the northern Han population and the local population through marriage.

To improve the accuracy of risk estimates for genetic counseling (Letra et al., 2007), we also stratified all cleft cases into the subgroups CLP and CL according to the clinical characteristics and pathogenesis (Spritz, 2001). However, such stratification to obtain more appropriate homogeneous subgroups also resulted in lower study power for these analyses. For our analysis of rs987525 we retained 96% statistical power (dominant model) or 98% statistical power (log additive model) to detect the heterogeneous OR of 2.57 observed in German samples (Birnbaum et al., 2009) of the NSCL/P group compared with controls. Nevertheless, we found that the frequencies of rs987525 minor allele A in our case, parent, and control populations were considerably low and distributions of alleles and genotypes were very similar in all our study groups. The frequency of the rs987525 A allele in the control group was 6.5%; however, previous studies found this frequency to be 20%–30% in European (Birnbaum et al., 2009; Grant et al., 2009) and about 12% in Mesoamerican (Rojas-Martinez et al., 2010) populations (Birnbaum et al., 2009; Grant et al., 2009). The lack of association between 8q24 and NSCL/P has also been reported in the American Hispanic population (Blanton et al., 2010). It is therefore possible that these variations may be explained by differences in ethnic background. Given the low frequencies of rs987525 A allele in our subjects, it is unlikely that this polymorphism plays a key role in the pathogenesis of NSCL/P in the southern Han Chinese located in Guangdong.

Our study has several unique strengths. First, we systematically studied the association between rs987525 and the risk of NSCL/P using family-based and case–control approaches. Such joint analyses can accurately estimate genetic effects in our study population, even when using an ascertained family sample. Second, our study population, both cases and controls, was quite homogeneous with respect to social and cultural norms compared with the larger groups of Asians and all Han Chinese from different provinces previously studied together, so our analyses will provide better isolation of the genetic risk in Guangdong. To further avoid any potential biases due to population admixture/stratification, misspecification of the trait distribution, or selection based on trait, we conducted TDT and FBAT. Our findings from a total of 601 individuals within 208 families further confirmed our results from the case–control study.

Overall, our data do not confirm the susceptibility role of 8q24 in NSCL/P that has been demonstrated in other GWAS and case–control association studies in other populations (Birnbaum et al., 2009; Grant et al., 2009; Beaty et al., 2010; Blanton et al., 2010; Rojas-Martinez et al., 2010). This is unlikely due to population stratification because our family-based association study design avoids concerns due to confounding or population stratification between the cases and unrelated controls (Beaty et al., 2002). Given the etiology of NSCL/P is complex and heterogeneous (Mossey and Little, 2009), the lack of association between 8q24 and the susceptibility of NSCL/P in our study population of southern Han Chinese located in Guangdong is unsurprising and further emphasizes the importance of recognizing geographical differences in ethnic origin within such studies.

Footnotes

Acknowledgments

The authors would like to thank all participating individuals and their families. The authors also sincerely thank Dr. Frieda Law and Dr. William Ba-Thein for their kind assistance during the preparation of this manuscript. This research was supported by the Likashing Foundation.

Disclosure Statement

The authors declare that they have no competing interests.

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