Abstract
Objective
Recent genome-wide association studies identified susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL±P) on 8q24.21, 10q25.3, 13q31.1, 15q13.3, 17q22, and 18q22 in populations of European origin. The purpose of this study was to determine, using DNA samples, whether 8q24.21 was a susceptibility locus for the development of NSCL±P in Japanese patients.
Methods
We used DNA from 167 Japanese NSCL±P patients (45 cleft lip without cleft palate and 122 cleft lip with cleft palate patients) and 190 Japanese unaffected control individuals. We performed an association study using 13 single nucleotide polymorphisms (SNPs) selected on the 8q24.21 locus. Genotyping of each SNP was carried out by direct sequencing of genomic DNA. Additionally, a haplotype block was constructed using the selected SNPs.
Results
The 13 selected SNPs were successfully genotyped in 357 individuals. The p values obtained were not low enough to indicate a significant association between the haplotypes and the development of NSCL±P in this population.
Conclusions
Our results suggest that the 8q24.21 locus is not associated with susceptibility to NSCL±P in Japanese patients and provide further evidence that ethnicity is a strong factor in determining susceptibility loci, albeit using a limited number of samples. Further studies are needed to identify regions involved in the development of NSCL±P in the Japanese population.
Cleft lip with or without cleft palate (CL±P) is one of the most common birth defects worldwide. Of variable phenotype, the incidence of this disease varies markedly depending on ethnicity, and the frequency of orofacial clefts in Japan is higher than that in other countries (Wyszynski et al., 1996; Murthy and Bhaskar, 2009).
CL±P comprises a heterogeneous group of congenital malformations and is typically classified into two categories: syndromic and nonsyndromic clefting. Genetic and environmental factors are involved in the pathogenesis of nonsyndromic cleft lip with or without cleft palate (NSCL±P). Much research into inherited factors for NSCL±P has been done in both animals and humans. Although many molecular genetic studies have identified genes or loci associated with CL±P, the underlying mechanism of this disease remains to be clarified (Murray and Schutte, 2004; Carinci et al., 2007; Jugessur et al., 2009; Marazita et al., 2009; Murthy and Bhaskar, 2009). Genome-wide association studies (GWAS) aim at identifying unknown disease factors not found by linkage analysis with existing loci and thus determining phenotypical correlations with various genotypes (Murthy and Bhaskar, 2009).
Recent GWAS have identified susceptibility loci for NSCL±P on 8q24.21, 10q25.3, 13q31.1, 15q13.3, and 17q22 in European (Birnbaum et al., 2009; Beaty et al., 2010; Mangold et al., 2010) and Mesoamerican populations (Rojas-Martinez et al., 2010). Another GWAS from Philadelphia also identified loci 8q24 and 18q22 for NSCL±P in individuals of European descent (Grant et al., 2009). Although few other studies have reported 8q24 as a locus in NSCL±P, this region has been well reported in epilepsy and tumor. The MYC enhancer region downstream of 8q24 was reported to be closely involved in the development of epilepsy (Zeidler et al., 1994; Morita et al., 1998; Wasserman et al., 2010). The aim of the present study was to determine whether 8q24, which showed a significant p value in two independent GWAS, was associated with susceptibility to NSCL±P in a Japanese population.
Materials and Methods
We used DNA collected from 167 Japanese NSCL±P patients (45 cleft lip without cleft palate patients and 122 cleft lip with cleft palate patients) at Nagasaki University Hospital and Tokyo Dental College Hospital. All diagnoses were based on examinations by well-trained plastic or oral surgeons. We also examined 190 Japanese unaffected adult control individuals at Nagasaki University Hospital. The study protocol was approved by the Committee for the Nagasaki University Ethical Committee on Human Genome and Gene Analysis.
Genotyping
We selected 13 single nucleotide polymorphisms (SNPs) (Table 1) located on 8q24.21 that were identified in a GWAS of Central European NSCL±P (Birnbaum et al., 2009). Among these, three SNPs (rs17241253, rs1530300, and rs987525) were reported to be the most significant (Birnbaum et al., 2009). The other 10 SNPs (rs11994831, rs7845615, rs1155582, rs1519851, rs10956449, rs17819888, rs1157136, rs10956450, rs6470670, and rs12548036) were chosen because their minor allele frequencies (MAF) were relatively high in the Japanese population. A search of the HapMap 3 database for the 13 SNPs targeted in this study in the Japanese yielded the following values: rs1530300, 0.012; rs987525, 0.042; and rs17241253, 0. Of the remaining SNPs, four were ~0.1 to ~0.3, while no data were available on the others. Genotyping of each SNP was carried out by direct sequencing of genomic DNA extracted from peripheral blood lymphocytes using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and primers designed from data at the University of California, Santa Cruz, Genome Browser Home (http://genome.ucsc.edu/). Polymerase chain reaction (PCR) was performed in 10 μL of reaction mixture with the DNA Thermal Cycler Model 9700 (Applied Biosystems, Applied Biosystems, Carlsbad, California, U.S.) under the appropriate conditions. The PCR products were subjected to Exonuclease I (Epicentre, Madison, WI) and shrimp alkaline phosphatase digestion (GE Healthcare UK Ltd., GE Healthcare UK Ltd., Little Chalfont, England) prior to sequencing. Direct sequencing was carried out using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems). Samples were purified with Sephadex G-50 (GE Healthcare UK Ltd., Little Chalfont, England) and electrophoresed on the Autosequencer Model 3130x1 (Applied Biosystems, Carlsbad, California, U.S.). The sequences were aligned with ATGC software (Genetyx Corp., Tokyo, Japan), and the genotypes of the SNPs were determined by visual inspection.
Association Between Japanese NSCL±P * and Single Nucleotide Polymorphisms (SNPs) at 8q24.21
NSCL±P = nonsyndromic cleft lip with or without palate; MAF = minor allele frequency; OR = odds ratio; CI = confidence interval.
574 samples were analyzed for estimation of allele frequency.
Statistical Analysis
All statistical tests for association were carried out using PLINK (Purcell et al., 2007) and Haploview software (Barrett et al., 2005). We tested for deviation from the Hardy-Weinberg equilibrium and for association with the Cochran-Armitage trend test. The odds ratio (OR) and corresponding 95% confidence interval for each SNP were also calculated.
Results and Discussion
The 13 selected SNPs were successfully genotyped in 357 individuals. No significant departures from the Hardy-Weinberg equilibrium were observed (all Hardy-Weinberg equilibria, p > 0.1). The results of the association analysis are shown in Table 1. For all 13 SNPs, the p values were above .1. The MAF of SNP rs17241253 was 0 in our analysis; therefore, neither a p value nor an OR could be calculated. Association mapping was performed, the linkage disequilibrium (LD) structure determined, and p values of haplotypes calculated with Haploview (Fig. 1, Table 2). SNP rs17241253 showed the highest OR in an earlier study by Birnbaum et al. (2009). However, a search of the HapMap database revealed that the MAF for rs17241253 was very low in the Japanese population. Although there is a strong correlation between the development of CL±P and a lower MAF value for rs17241253 in Europeans, only the TT homozygous genotype was found here, making this SNP not appropriate for CLP studies in the Japanese population. Therefore, we selected SNPs near rs17241253 for our haplotype analysis. Haplotype was defined as r-square > 0.8, and an association test was then performed by Haploview. No haplotype block including rs17241253 showed a statistically significant p value in our single SNP or haplotype association analysis. Moreover, rs11994831, rs7845615, rs1530300, rs987525, and rs12548036 were not included in this haplotype block.
The linkage disequilibrium display was analyzed using Haploview software. Blocks show haplotypes constructed from the results of our analysis.
Haplotypes and the Result of Their Association Study *
LD Plot in Fig. 1 shows establishment of the combination between each haplotype.
We clearly verified that the LD block including rs17241253 was not associated with NSCL±P. The associated region reported by Birnbaum et al. (2009) was greater than 185 kb. This LD block including the associated region was divided into two definitive blocks in the European population. In contrast, very weak LD blocks were observed in Japanese and were not definitive when analyzed using the HapMap database (data not shown). The results of this study suggest that LD blocks including rs17241253 indicate no association with NSCL±P in the Japanese population. Our results suggest that rs1530300 and rs987525 are also not associated with NSCL±P in the Japanese population. This is similar to the results of Beaty et al. in other Asian populations (2010). These results indicate that an SNP may have a low association in Asians, even if it has a high association in Europeans.
The pathogenesis of NSCL±P is very complex, and both genetic and environmental factors are involved (Mossey et al., 2009). It is thought that many genes and proteins are involved in the pathogenesis of NSCL±P, and many genes and loci associated with NSCL±P have been reported (Beaty et al., 2010). While the results of this study provide no conclusive evidence of an association between specific loci and the development of NSCL±P in the Japanese population, the incidence of NSCL±P has been shown to vary depending on ethnicity: It is high in Asia and Latin America and low in Israel, South Africa, and Southern Europe (Mossey et al., 2009), for example. These differences may result from genetic background, and the Japanese population may have its own susceptibility gene or locus, such as MAFB on 20q12, IRF6 on 1q32.2, or ABCA4 on 1p22.1 (Beaty et al., 2010). These results suggest that it is difficult to apply findings from European populations to Asian populations. Further study is needed to identify regions involved in the development of NSCL±P in the Japanese population. The incidence of this disease is higher in Asian populations, making the identification of loci involved in its development all the more important.
Footnotes
Acknowledgments
We thank the families for their participation. We thank Ms. C. Hayashida and Ms. M. Ohga for their technical assistance. We would also like to thank Associate Professor Jeremy Williams, Tokyo Dental College, for his assistance with the English of the manuscript. This work was supported in part by grants from the Ministry of Health, Labour and Welfare (K.Y.), the Japan Society for the Promotion of Science (K.Y.: 21390100, A. Kinoshita: 20590331), the Takeda Scientific Foundation, and the Naito Foundation (K.Y.).