Case-parent trios were used in a genome wide association study of cleft lip with/without cleft palate (CL/P). SNPs near two genes not previously associated with CL/P [MAFB: most significant SNP rs13041247, with odds ratio per minor allele OR=0.704; 95%CI=0.635,0.778; p=2.05*10 −11 ; and ABCA4: most significant SNP rs560426, with OR=1.432; 95%CI=1.292,1.587; p=5.70*10 −12 ] and two previously identified regions (chr. 8q24 and IRF6) attained genome wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes were similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24 while Asian families showed stronger evidence for MAFB and ABCA4. Expression studies support a role for MAFB in palate development.Corresponding author: THB (tbeaty@jhsph.edu). NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 September 17. Published in final edited form as:Nat Genet. 2010 June ; 42(6): 525-529. doi:10.1038/ng.580. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCleft lip with or without cleft palate (CL/P) is a common human birth defect with documented genetic and environmental risk factors 1 . While CL/P can occur in many Mendelian malformation syndromes, the isolated, non-syndromic form constitutes 70% of all cases2. Evidence for genetic control of CL/P is compelling: recurrence risks are 20-30 times greater than population prevalences3 , 4 and both twin and family studies 5 suggest a major role for genes, Mutations in IRF6 cause VanderWoude syndrome, the most common Mendelian syndrome including CL/P, and markers in IRF6 have repeatedly shown evidence of association with isolated, non-syndromic CL/P 6-9 . An allele disrupting an AP2 binding site near IRF6 showed particularly strong evidence among European CL families, although multiple risk alleles are likely 10 .Birnbaum et al. 11 conducted a case-control genome wide association study (GWAS) in Germany and found significant evidence of association with markers in 8q24.21, and a US case-control GWAS confirmed this region 12 , with rs987525 being the most significant marker in both studies. Here we present a GWAS using a case-parent trio design in a consortium drawing cases from Europe, the US, China, Taiwan, Singapore, Korea and the Philippines. This design has the advantage of being robust to confounding due to population stratification, which is important when cases from diverse populations are combined. ResultsBecause these case-parent trios came from different populations (Table 1), we conducted a principal components analysis (PCA) on all parents to document genetic variation in our consortium (Supplementary Figure 1). Approximately 50% of parents could be classified as Asian and 45% as European, with remaining parents being of African or "other" ancestry (including mixed). Transmission disequilibrium tests...

Non-syndromic cleft palate (CP) is a common birth defect with a complex and heterogeneous etiology involving both genetic and environmental risk factors. We conducted a genome wide association study (GWAS) using 550 case-parent trios, ascertained through a CP case collected in an international consortium. Family based association tests of single nucleotide polymorphisms (SNP) and three common maternal exposures (maternal smoking, alcohol consumption and multivitamin supplementation) were used in a combined 2 df test for gene (G) and gene-environment (G×E) interaction simultaneously, plus a separate 1 df test for G×E interaction alone. Conditional logistic regression models were used to estimate effects on risk to exposed and unexposed children. While no SNP achieved genome wide significance when considered alone, markers in several genes attained or approached genome wide significance when G×E interaction was included. Among these, MLLT3 and SMC2 on chromosome 9 showed multiple SNPs resulting in increased risk if the mother consumed alcohol during the peri-conceptual period (3 months prior to conception through the first trimester). TBK1 on chr. 12 and ZNF236 on chr. 18 showed multiple SNPs associated with higher risk of CP in the presence of maternal smoking. Additional evidence of reduced risk due to G×E interaction in the presence of multivitamin supplementation was observed for SNPs in BAALC on chr. 8. These results emphasize the need to consider G×E interaction when searching for genes influencing risk to complex and heterogeneous disorders, such as non-syndromic CP.

Purpose:The interferon regulatory factor 6 (IRF6), the gene that causes van der Woude syndrome has been shown to be associated with nonsyndromic cleft lip with or without palate in several populations. This study aimed to confirm the contribution of IRF6 to cleft lip with or without palate risk in additional Asian populations. Methods: A set of 13 single nucleotide polymorphisms was tested for association with cleft lip with or without palate in 77European American, 146 Taiwanese, 34 Singaporean, and 40 Korean case-parent trios using both the transmission disequilibrium test and conditional logistic regression models. Results: Evidence of linkage and association was observed among all four populations; and two specific haplotypes [GC composed of rs2235373-rs2235371 (p.V274I) and AAG of rs599021-rs2235373-rs595918] showed the most significant over-and undertransmission among Taiwanese cases (P ϭ 9 ϫ 10 Ϫ6 and P ϭ 5 ϫ 10 Ϫ6 , respectively). The AGC/CGC diplotype composed of rs599021-rs2235373-rs2013162 showed almost a 7-fold increase in risk among the Taiwanese sample (P Ͻ

Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence of 1 in 700 live births. The paired box (PAX) genes have been suggested as candidate genes for CL/P based largely on mouse models; however, few human studies have focused on this gene family. This study tests for association between markers in four PAX genes and CL/P using a case-parent trio design considering parent-of-origin effects. Trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 34 single nucleotide polymorphisms (SNPs) in the PAX3, PAX6, PAX7, and PAX9 genes. We performed the transmission disequilibrium test (TDT) on individual SNPs. Parent-of-origin effects were assessed using the transmission asymmetry test (TAT) and the parent-of- origin likelihood ratio test (PO-LRT). TDT analysis showed one SNP (rs766325) in PAX7 yielding evidence of linkage and association when parent-of-origin was not considered, with an OR(transmission) = 1.62 (P = 0.003), and five SNPs in PAX6 (including two pairs in near perfect linkage disequilibrium). TAT analysis of all trios revealed two SNPs in PAX7 and four SNPs in PAX3 showing significant excess maternal transmission. For these six SNPs, the maternal OR(transmission) ranged between 1.74 and 2.40, and PO-LRT was also significant (P-values = 0.035–0.012). When this analysis was limited to trios with male cases, SNPs in PAX7 showed higher maternal OR(transmission) and greater significance. PAX genes may influence the risk of CL/P through maternal effects, possibly imprinting, which seems to be stronger among male cases.

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.

Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence around 1 in 700 live births. The Runt-related transcription factor 2 (RUNX2) gene has been suggested as a candidate gene for CL/P based largely on mouse models; however, no human studies have focused on RUNX2 as a risk factor for CL/P. This study examines the association between markers in RUNX2 and isolated, nonsyndromic CL/P using a case-parent trio design, while considering parent-of-origin effects. Case-parent trios from four populations (77 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 24 single nucleotide polymorphisms (SNPs) in the RUNX2 gene. We performed the transmission disequilibrium test on individual SNPs. Parent-of-origin effects were assessed using the transmission asymmetry test and the parent-of-origin likelihood ratio test (PO-LRT). When all trios were combined, the transmission asymmetry test revealed a block of 11 SNPs showing excess maternal transmission significant at the P < 0.01 level, plus one SNP (rs1934328) showing excess paternal transmission (P = 0.002). For the 11 SNPs showing excess maternal transmission, odds ratios of being transmitted to the case from the mother ranged between 3.00 and 4.00. The parent-of-origin likelihood ratio tests for equality of maternal and paternal transmission were significant for three individual SNPs (rs910586, rs2819861, and rs1934328). Thus, RUNX2 appears to influence risk of CL/P through a parent-of-origin effect with excess maternal transmission.

The International Task Force on Volunteer Cleft Missions was set up to provide a report to be presented at the Eighth International Congress of Cleft Palate and Associated Craniofacial Anomalies on September 12, 1997, in Singapore. The aim of the report was to provide data from a wide range of different international teams performing volunteer cleft missions and, thereafter, based on the collected data, to identify common goals and aims of such missions. Thirteen different groups actively participating in volunteer cleft missions worldwide were selected from the International Confederation of Plastic and Reconstructive Surgery's list of teams actively participating in volunteer cleft missions. Because of the time frame within which the committee had to work, three groups that did not respond by the stipulated deadline were omitted from the committee. The represented members and their respective institutions have undertaken more than 50 volunteer cleft missions to underdeveloped nations worldwide within the last 3 years. They have visited over 20 different countries, treating more than 3,500 patients worldwide. Based on the data collected and by consensus, the committee outlined recommendations for future volunteer cleft missions based on 1) mission objectives, 2) organization, 3) personal health and liability, 4) funding, 5) trainees in volunteer cleft missions, and 6) public relations. The task force believed that all volunteer cleft missions should have well-defined objectives, preferably with long-term plans. The task force also decided that it was impossible to achieve a successful mission without good organization and close coordination. All efforts should be made, and care taken, to ensure that there is minimal morbidity and no mortality. Finally, as ambassadors of goodwill and humanitarian aid, the participants must make every effort to understand and respect local customs and protocol. The main aims are to provide top-quality surgical service, train local doctors and staff, develop and nurture fledgling cleft programs, and, finally, make new friends.

This study examined the association between markers in transforming growth factor alpha (TGFA) and isolated, non-syndromic cleft lip with/without palate (CL/P) using a case-parent trio design, considering parent-of-origin effects. We also tested for gene-environmental interaction with common maternal exposures, and for gene-gene interaction using markers in TGFA and another recognized causal gene, IRF6. CL/P case-parent trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 17 single nucleotide polymorphisms (SNPs) in TGFA. The transmission disequilibrium test was used to test individual SNPs, and the parent-of-origin likelihood ratio test (PO-LRT) was used to assess parent-of-origin effects. We also screened for possible gene-environment interaction using PBAT, and tested for gene-gene interaction using conditional logistic regression models. When all trios were combined, four SNPs showed significant excess maternal transmission, two of which gave significant PO-LRT values [rs3821261: P = 0.004 and OR(imprinting) = 4.17; and rs3771475: P = 0.027 and OR (imprinting) = 2.44]. Haplotype analysis of these two SNPS also supported excess maternal transmission. We saw intriguing but suggestive evidence of G × E interaction for several SNPs in TGFA when either individual SNPs or haplotypes of adjacent SNPs were considered. Thus, TGFA appears to influence risk of CL/P through unconventional means with an apparent parent-of-origin effect (excess maternal transmission) and possible interaction with maternal exposures.