Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Non-syndromic (NS) cleft lip with or without cleft palate (CL/P) is a common disorder with a strong genetic underpinning. Genome-wide association studies have detected common variants associated with this disorder, but a large portion of the genetic risk for NSCL/P is conferred by unidentified rare sequence variants. Mutations in IRF6 (Interferon Regulatory Factor 6) and GRHL3 (Grainyhead-like 3) cause Van der Woude syndrome, which includes CL/P. Both genes encode members of a regulatory network governing periderm differentiation in model organisms. Here, we report that Krüppel-like factor 17 (Klf17), like Grhl3, acts downstream of Irf6 in this network in zebrafish periderm. Although Klf17 expression is absent from mammalian oral epithelium, a close homologue, Klf4, is expressed in this tissue and is required for the differentiation of epidermis. Chromosome configuration capture and reporter assays indicated that IRF6 directly regulates an oral-epithelium enhancer of KLF4. To test whether rare missense variants of KLF4 contribute risk for NSCL/P, we sequenced KLF4 in approximately 1000 NSCL/P cases and 300 controls. By one statistical test, missense variants of KLF4 as a group were enriched in cases versus controls. Moreover, two patient-derived KLF4 variants disrupted periderm differentiation upon forced expression in zebrafish embryos, suggesting that they have dominant-negative effect. These results indicate that rare NSCL/P risk variants can be found in members of the gene regulatory network governing periderm differentiation.

Type II (non-insulin-dependent) diabetes mellitus is a common disorder affecting more than 100 million people throughout the world. In most cases it shows a strong genetic component, particularly in families with an earlier onset of the disease. Apart from the successful identification of the underlying genetic defects in the relatively rare monogenic forms of maturity-onset diabetes in the young (MODY) little success has been made so far in the dissection of the genetic causes of the presumably polygenic forms of late-onset Type II diabetes. To date five genes have been associated with MODY, i. e. hepatocyte nuclear factor (HNF 4a (MODY1), glucokinase (GCK) (MODY2), the HNF-1a gene (MODY3), insulin pro- Diabetologia (2001) Abstract Aims/hypothesis. The aim of this study was to examine the putative role of mutations in the insulin promoter 1 (IPF1) gene in early-onset diabetes. Methods. We carried out mutation screening of the IPF1 gene in 115 Scandinavian families with at least two members with onset of diabetes younger than 40 years. The allele frequencies were also tested in 183 unrelated patients with late-onset Type II (noninsulin-dependent) diabetes mellitus and in 92 nondiabetic control subjects. Results. Two novel IPF1 variants (G212R and P239Q) and one previously reported (D76N) IPF1 variant were identified in the 115 families (3.5 %). The D76N variant was found in one MODY3 family (S315fsinsA of HNF1a) and also in two families with late-onset Type II diabetes. The P239Q variant was identified in two families with early-onset diabetes including one with MODY3 (R272C of HNF 1a) and in three families with late-onset Type II diabetes. Despite the fact that the variants did not segregate completely with diabetes, the non-diabetic carriers of the IPF1 variants had increased blood glucose concentrations (p < 0.05) and reduced insulin:glucose ratios (p < 0.05) during an oral glucose tolerance test compared with non-diabetic family members without these variants. In addition, when the G212R and P239Q variants were expressed in cells without IPF1 i. e. . Nes2 y cells, both variants showed about a 50 % reduction in their ability to activate insulin gene transcription compared to wild-type IPF1, as measured by reporter gene assay. Conclusion/interpretation. Although mutations in the IPF-1 gene are rare in early-(3.5 %) and late-onset (2.7 %) Type II diabetes, they are functionally important and occur also in families with other MODY mutations. [Diabetologia (2001) 44: 249±258]

A film paradigm was developed to examine baseline and emotion modulated startle across a broad age range from preschool to adulthood. The paradigm was tested in children (3-, 5-, 7- and 9-year-olds) and adults (total N =122). The paradigm elicited a similar startle potentiation pattern across age groups; however, baseline startle changed with age: 3- and 5-year olds showed lower response probability and magnitude of baseline startle than adults. Females exhibited larger baseline startle response probability and overall magnitude than did males; however, no sex by emotion modulated startle interaction was noted. Anxiety measures were obtained for all children. Individual differences in anxiety were associated with baseline startle magnitude among older but not younger children. No association of anxiety with startle potentiation was noted. Overall the film paradigm was applicable across a wide developmental span, revealing potential developmental and gender differences in baseline startle magnitude and response probability.

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