Stefanie Birnbaum

Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24.

We conducted a genome-wide association study involving 224 cases and 383 controls of Central European origin to identify susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). A 640-kb region at chromosome 8q24.21 was found to contain multiple markers with highly significant evidence for association with the cleft phenotype, including three markers that reached genome-wide significance. The 640-kb cleft-associated region was saturated with 146 SNP markers and then analyzed in our entire NSCL/P sample of 462 unrelated cases and 954 controls. In the entire sample, the most significant SNP (rs987525) had a P value of 3.34 × 10−24. The odds ratio was 2.57 (95% CI = 2.02–3.26) for the heterozygous genotype and 6.05 (95% CI = 3.88–9.43) for the homozygous genotype. The calculated population attributable risk for this marker is 0.41, suggesting that this study has identified a major susceptibility locus for NSCL/P.

Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate

We conducted a genome-wide association study for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in 401 affected individuals and 1,323 controls, with replication in an independent sample of 793 NSCL/P triads. We report two new loci associated with NSCL/P at 17q22 (rs227731, combined P = 1.07 × 10−8, relative risk in homozygotes = 1.84, 95% CI 1.34–2.53) and 10q25.3 (rs7078160, combined P = 1.92 × 10−8, relative risk in homozygotes = 2.17, 95% CI 1.32–3.56).

IRF6 gene variants in Central European patients with non-syndromic cleft lip with or without cleft palate

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non-syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case-control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non-synonymous coding variant V274I (rs2235371) and five IRF6-haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 x 10(-6)) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38-2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21-3.10) for the homozygous genotype, values that are similar to those reported in a previously published family-based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP-based and resequencing studies using large samples of patients.

Genome-wide linkage scan of nonsyndromic orofacial clefting in 91 families of central European origin†

Orofacial clefts are among the most common of all congenital disorders. Nonsyndromic cases of cleft lip with or without cleft palate (NSCL/P) and cleft palate only (NSCPO) are considered to have a multifactorial etiology which involves both genetic and environmental factors. We present the results of a genome‐wide linkage scan in 91 families of central European descent with nonsyndromic orofacial clefts (NSC). The sample included 74 NSCL/P families, 15 NSCPO families, and 2 mixed families (a total of 217 affected and 230 unaffected individuals were genotyped). We genotyped 542 microsatellite markers (average intermarker distance = 6.9 cM). Multipoint nonparametric linkage analysis was performed using Allegro 2.0f. In addition to the factors investigated in previous genome‐wide linkage analyses, we searched for sex‐specific susceptibility loci, loci demonstrating parental imprinting and loci that are shared by NSCL/P and NSCPO. Several genomic regions likely to contain susceptibility loci for NSC were identified at the level of nominal significance. Some of these overlap with regions identified in previous studies. Suggestive evidence of linkage was obtained for the loci 4q21‐q26 and 1p31‐p21, with the chromosome 1 locus showing a male‐specific genetic effect. Our study has identified promising chromosomal regions for the identification of NSC‐associated genes, and demonstrates the importance of performing detailed statistical analyses which take into account complex genetic mechanisms such as sex‐specific effects and genomic imprinting. Further research in large patient samples is necessary to identify factors common to NSCL/P and NSCPO.

TGFB3 displays parent-of-origin effects among central Europeans with nonsyndromic cleft lip and palate

AbstractMice with a deletion of Tgf-β3 (−/−) and association studies in humans of different ethnicities support the involvement of TGFB3 in the etiology of orofacial clefts. In this study, we investigated the relevance of TGFB3 in the development of cleft lip and palate (CL/P) among 204 triads of central European origin. Transmission-disequilibrium test (TDT) analysis revealed no significant transmission distortions for each marker alone, and none for any possible haplotypes. However, we found strong evidence for parent-of-origin effects, with lower risk of maternal transmission compared with paternal transmission [IM = 0.38; confidence interval (CI): 0.17–0.86] of the risk allele T to an affected offspring at marker rs2300607. This is also expressed in an increased risk of heterozygous children having the T allele inherited from the father (RP = 3.47; CI: 1.32–9.11). Our data support the involvement of TGFB3 in the development of oral clefts in patients of central European origin.

Replication of Novel Susceptibility Locus for Nonsyndromic Cleft Lip With or Without Cleft Palate on Chromosome 8q24 in Estonian and Lithuanian Patients

Cleft lip with or without cleft palate (CL/P) is one of the mostcommonhumanbirthdefects.CL/Pmayoccuraspartofacomplexmalformation syndrome or, as is more commonly the case, as anonsyndromic malformation. In European populations non-syndromic cleft lip with or without cleft palate (NSCL/P) has abirth prevalence of 1/700–1/1,000. NSCL/P has a multifactorialetiology resulting from interactions between multiple genetic andenvironmentalfactors.Nonsyndromiccleftpalateonly(NSCPO)isthe second most common form of nonsyndromic clefting, with aprevalence of 1/2,000 in most European populations. AlthoughNSCL/P and NSCPO are generally considered to be develop-mentally and genetically distinct entities, there is evidence thatthey may have an etiological overlap [Kondo et al., 2002; Sivertsenet al., 2008].The identification of susceptibility genes for NSCL/P has beenthe subject of extensive research. Of the many candidate genesinvestigated, however, only the IRF6 gene has shown a convincingdegreeofconsistencyacrossstudies[Zuccheroetal.,2004;Rahimovet al., 2008]. The first genome-wide association scan (GWAS)recently reported a new major susceptibility locus at chromosome8q24.21,with the strongestassociationsignal having been observedfor rs987525 (P¼3.34 10

Mutation Screening in the IRF6-Gene in Patients With Apparently Nonsyndromic Orofacial Clefts and a Positive Family History Suggestive of Autosomal-Dominant Inheritance

Stefanie Birnbaum,* Heiko Reutter, Carola Lauster, Martin Scheer, Gül Schmidt, Mitra Saffar, Markus Martini, Alexander Hemprich, Henning Henschke, Franz-Josef Kramer, and Elisabeth Mangold Institute of Human Genetics, University of Bonn, Bonn, Germany Department of Cleft Lip and Cleft Palate Surgery, Humboldt University of Berlin, Berlin, Germany Department of Oral and Maxillo-Facial Surgery, University of Cologne, Köln, Germany Department of Orthodontics, University of Cologne, Köln, Germany Department of Oral and Maxillo-Facial Surgery, University of Bonn, Bonn, Germany Department of Oral and Maxillo-Facial Surgery, University of Leipzig, Leipzig, Germany Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany Department of Oral and Maxillo-Facial Surgery, University of Göttingen, Göttingen, Germany

Susceptibility locus for non-syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental factors. Recently, two novel susceptibility loci and three suggestive loci for NSCL/P were identified by a genome-wide association scan (GWAS) in a German population with subsequent independent replication in a mixed European population. The aim of the present study was to investigate whether these newly detected loci confer similar effects in the North-East European Baltic population. A total of 101 NSCL/P patients and 254 controls from Estonia were included. A significant association was observed for rs7078160 (P = 0.0016) at chromosome 10q25, which confirms the association of this locus with NSCL/P in the Baltic population. No significant association was found for the other four loci, a result that may have been attributable to the limited power of the sample.

Family-based association study of the MTHFR polymorphism C677T in patients with nonsyndromic cleft lip and palate from central Europe.

Objective: The 677C→T allele in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene has been implicated in the etiology of nonsyndromic cleft lip and palate (CL/P). This study involved a family-based association study of the MTHFR polymorphism. Patients/participants: We examined 181 patients with CL/P of central European descent and their parents for this variant. Results: The transmission disequilibrium test (TDT) did not confirm an association between the MTHFR 677C→T polymorphism and nonsyndromic CL/P as previously suggested (p = .36). When comparing the offspring of mothers with periconceptional use of folate to those without, no statistically significant differences were found (p = .708). Conclusion: Our data suggest that the MTHFR 677C→T polymorphism does not make a major contribution to the occurrence of CL/P among central Europeans.

Further evidence for the involvement of MYH9 in the etiology of non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental components. MYH9, the gene coding for the heavy chain of non-muscle myosin II, has been considered as a good candidate gene in NSCL/P on the basis of its expression profile during craniofacial morphogenesis. Reports in an Italian sample, as well as in an ethnically mixed North American sample, of a positive association between single-nucleotide polymorphisms in the MYH9 gene and NSCL/P have provided further support for the role of MYH9 in the development of NSCL/P. In the present study, we aimed to replicate these findings by conducting a family-based association study with seven single nucleotide polymorphisms in MYH9 using a sample of 248 NSCL/P patients and their parents. Single marker analysis resulted in a highly significant association for rs7078. In haplotype analysis, the most significant result was obtained for the SNP combination (rs7078; rs2071731; rs739097; rs5995288). Our results thus confirm the potential involvement of MYH9 in the etiology of NSCL/P in our patients of Central European origin, although further studies are warranted to determine its exact pathogenetic role.