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...

DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Definition: ECLAMC (‘Estudio Colaborativo Latino Americano de Malformaciones Congénitas’) is a program for the clinical and epidemiological investigation of risk factors in the etiology of congenital anomalies in Latin-American hospitals, using a case-control methodological approach. It is a voluntary agreement among professionals lacking institutional base as well as designated budgets. ECLAMC has been usually funded by research-funding agencies rather than public health ministries. The National Research Councils of Argentina and Brazil have been the main sources of support during its 36 years of existence. Since vital and health statistics are unreliable in South America, ECLAMC collects all the information required for the denominators in a hospital-based sample of births. ECLAMC can be defined as a continental network of persons interested in research and prevention of birth defects. History and Evolution: From the institutional point of view, ECLAMC has had headquarters in diverse centers of Argentina and Brazil, but always as an independent research project, without a defined administrative link. ECLAMC began operating in 1967, as an investigation limited to the city of Buenos Aires, Argentina, and it gradually expanded until covering all the 10 countries of South America as well as Costa Rica and the Dominican Republic. Even though ECLAMC has maintained essentially the same original experimental design since 1967, due to the data accumulated by the program, the increasing experience as well as the development in science, technical modifications occurred including a DNA bank and a fully informatized data handling system. Since 1974 ECLAMC has been a founder member of the International Clearinghouse for Birth Defects Monitoring Systems; since 1994 a WHO Collaborating Center for the Prevention of Congenital Malformations, and since 2000 a collaborating member of the NIH Global Netwok for Women’s and Children’s Health Research. Methodology: The maternity hospital network of ECLAMC examines around 200,000 births per year. All major and minor anomalies diagnosed at birth in infants weighing 500 g or more are registered according to a manual of procedures. The next non-malformed baby of the same sex born in the same hospital is selected as a control subject for each case. Thus, a one-to-one healthy control group matched by sex, time and place of birth is obtained. As a system of epidemic surveillance, ECLAMC systematically observes the fluctuations in the frequencies of different malformations and, in the case of an alarm for a probable epidemic of a given malformation, at a given moment, and given area, it acts to identify its cause. As termination of pregnancy has severe legal restrictions in South America, prevention of birth defects should concentrate on primary, preconceptional and tertiary measures. Tertiary measures aim to avoid complications of the affected patients from the medical, psychological, and social standpoints.

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.

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The spectrum of the VATER association has been debated ever since its description more than two decades ago. To assess the spectrum of congenital anomalies associated with VATER while minimizing the distortions due to small samples and referral patterns typical of clinical series, we studied infants with VATER association reported to the combined registry of infants with multiple congenital anomalies from 17 birth defects registries worldwide that are part of the International Clearinghouse for Birth Defects Monitoring Systems (ICB-DMS). Among approximately 10 million infants born from 1983 through 1991, the ICB-DMS registered 2,295 infants with 3 or more of 25 unrelated major congenital anomalies of unknown cause. Of these infants, 286 had the VATER association, defined as at least three of the five VATER anomalies (vertebral defects, anal atresia, esophageal atresia, renal defects, and radial-ray limb deficiency), when we expected 219 (P<0.001). Of these 286 infants, 51 had at least four VATER anomalies, and 8 had all five anomalies. We found that preaxial but not other limb anomalies were significantly associated with any combination of the four nonlimb VATER anomalies (P<0.001). Of the 286 infants with VATER association, 214 (74.8%) had additional defects. Genital defects, cardiovascular anomalies, and small intestinal atresias were positively associated with VATER association (P<0.001). Infants with VATER association that included both renal anomalies and anorectal atresia were significantly more likely to have genital defects. Finally, a subset of infants with VATER association also had defects described in other associations, including diaphragmatic defects, oral clefts, bladder exstrophy, omphalocele, and neural tube defects. These results offer evidence for the specificity of the VATER association, suggest the existence of distinct subsets within the association, and raise the question of a common pathway for patterns of VATER and other types of defects in at least a subset of infants with multiple congenital anomalies.

Nonsyndromic orofacial clefts (OFCs) are a heterogeneous group of common craniofacial birth defects with complex etiologies that include genetic and environmental risk factors. OFCs are commonly categorized as cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which have historically been analyzed as distinct entities. Genes for both CL/P and CP have been identified via multiple genome-wide linkage and association studies (GWAS), however, altogether, known variants account for a minority of the estimated heritability in risk to these craniofacial birth defects. We performed genome-wide meta-analyses of CL/P, CP, and all OFCs across two large, multiethnic studies. We then performed population specific meta-analyses in sub-samples of Asian and European ancestry. In addition to observing associations with known variants, we identified a novel genome-wide significant association between SNPs located in an intronic TP63 enhancer and CL/P (p = 1.16 × 10−8). Several novel loci with compelling candidate genes approached genome-wide significance on 4q21.1 (SHROOM3), 12q13.13 (KRT18), and 8p21 (NRG1). In the analysis of all OFCs combined, SNPs near FOXE1 reached genome-wide significance (p = 1.33 × 10−9). Our results support the highly heterogeneous nature of OFCs and illustrate the utility of meta-analysis for discovering new genetic risk factors.