Genome-wide association study of primary dentition pit-and-fissure and smooth surface caries.

Dental caries continues to be the most common chronic disease in children today. Despite the substantial involvement of genetics in the process of caries development, the specific genes contributing to dental caries remain largely unknown. We performed separate genome-wide association studies of smooth and pit-and-fissure tooth surface caries experience in the primary dentitions of self-reported white children in two samples from Iowa and rural Appalachia. In total, 1,006 children (ages 3-12 years) were included for smooth surface analysis, and 979 children (ages 4-14 years) for pit-and-fissure surface analysis. Associations were tested for more than 1.2 million single nucleotide polymorphisms, either genotyped or imputed. We detected genome-wide significant signals in KPNA4 (p value = 2.0E-9), and suggestive signals in ITGAL (p value = 2.1E-7) and PLUNC family genes (p value = 2.0E-6), thus nominating these novel loci as putative caries susceptibility genes. We also replicated associations observed in previous studies for MPPED2 (p value = 6.9E-6), AJAP1 (p value = 1.6E-6) and RPS6KA2 (p value = 7.3E-6). Replication of these associations in additional samples, as well as experimental studies to determine the biological functions of associated genetic variants, are warranted. Ultimately, efforts such as this may lead to a better understanding of caries etiology, and could eventually facilitate the development of new interventions and preventive measures.

Mutations in a new gene in Ellis-van Creveld syndrome and Weyers acrodental dysostosis.

Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly and dysplastic nails and teeth. Congenital cardiac defects, most commonly a defect of primary atrial septation producing a common atrium, occur in 60% of affected individuals. The disease was mapped to chromosome 4p16 in nine Amish subpedigrees and single pedigrees from Mexico, Ecuador and Brazil. Weyers acrodental dysostosis (MIM 193530), an autosomal dominant disorder with a similar but milder phenotype, has been mapped in a single pedigree to an area including the EvC critical region. We have identified a new gene (EVC), encoding a 992-amino-acid protein, that is mutated in individuals with EvC. We identified a splice-donor change in an Amish pedigree and six truncating mutations and a single amino acid deletion in seven pedigrees. The heterozygous carriers of these mutations did not manifest features of EvC. We found two heterozygous missense mutations associated with a phenotype, one in a man with Weyers acrodental dysostosis and another in a father and his daughter, who both have the heart defect characteristic of EvC and polydactyly, but not short stature. We suggest that EvC and Weyers acrodental dysostosis are allelic conditions.

Extending the spectrum of Ellis van Creveld syndrome: a large family with a mild mutation in the EVC gene.

BACKGROUND: Ellis-van Creveld (EvC) syndrome is characterized by short limbs, short ribs, postaxial polydactyly, dysplastic nails and teeth and is inherited in an autosomal recessive pattern. We report a family with complex septal cardiac defects, rhizomelic limb shortening, and polydactyly, without the typical lip, dental, and nail abnormalities of EvC. The phenotype was inherited in an autosomal recessive pattern, with one instance of pseudodominant inheritance. METHODS: Because of the phenotypic overlap with EvC, microsatellite markers were used to test for linkage to the EVC/EVC2 locus. The results did not exclude linkage, so samples were sequenced for mutations. RESULTS: We identified a c.1868T>C mutation in EVC, which predicts p.L623P, and was homozygous in affected individuals. CONCLUSION: We conclude that this EVC mutation is hypomorphic and that such mutations can cause a phenotype of cardiac and limb defects that is less severe than typical EvC. EVC mutation analysis should be considered in patients with cardiac and limb malformations, even if they do not manifest typical EvC syndrome.

Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions.

The basic helix-loop-helix domain (bHLH) is present in a large class of transcriptional regulators involved in developmental processes and oncogenesis. It determines DNA binding and specific homo- and heterodimeric protein associations, crucial for protein function. Myc and Max belong to a subset of HLH proteins, containing a leucine zipper (LZ) adjacent to the bHLH domain. They differ in dimerization and functional properties such as DNA binding and transcriptional activation, and their association is required for malignant transformation by Myc. To analyze the interaction specificity of Myc and Max bHLH-LZ domains, we developed a simple Escherichia coli genetic system, which uses the amino-terminal lambda phage cI repressor as a reporter for dimerization and allows an easy detection of dimeric interactions. By reciprocal exchanges of different Myc and Max subdomains (helix 1, helix 2 and leucine zipper), we showed that the recognition specificity of Max homodimers as well as of Myc/Max heterodimers is entirely determined by the helix 2-leucine zipper region, the major role being played by the leucine zipper. The Myc LZ was found to prevent homodimeric interactions, thus explaining Myc inability to homodimerize efficiently. Moreover, we showed that the system is valid as well for reproducing the interaction of HLH proteins not containing a leucine zipper and that the chimerical proteins maintain sequence-specific DNA binding.

Segmentally restricted, cephalic expression of a leucine zipper gene during Drosophila embryogenesis.

The expression pattern and DNA sequence of a newly identified gene, CNC (cap'n'collar), suggest a role for this gene in cephalic patterning during Drosophila embryogenesis. In situ hybridization reveals transcripts localized to the mandibular segment and the hypopharyngeal and labral primordia first detectable in late blastoderm stages. Sequence analysis of cDNA clones from the CNC locus shows the CNC gene product to be related to transcription factors of the leucine zipper (bZIP) class. Based on its protein sequence, we propose that CNC is a subunit of a heterodimeric regulatory protein involved in the control of head morphogenesis.

Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.

The leucine zipper transcription factor TSC-22 (TGF-beta1 Stimulated Clone-22) was first isolated from a mouse osteoblast cell line as an immediate-early target gene of TGF-beta1. However, work with other cell lines, as well as with a Drosophila homolog, bunched, suggests that it is an effector gene of various growth factors and potentially involved in the integration of multiple extracellular signals. Throughout mouse embryogenesis TSC-22 is expressed in a dynamic pattern. Although early TSC-22 expression is ubiquitous in 6.5 day embryos, as development proceeds TSC-22 expression is upregulated at sites of epithelial-mesenchymal interactions such as the limb bud, tooth primordiurn, hair follicle, kidney, lung, and pancreas. TSC-22 is also expressed in many neural crest-derived tissues including the mesenchyme of the branchial arches, the cranial, dorsal root, and sympathetic ganglia, as well as the facial cartilage and bone. Other areas of expression are the otic and optic vesicles, the heart, and cartilage and bone forming regions throughout the embryo.

Crystallization and preliminary X-ray crystallographic studies of nonhistone region of macroH2A.1.

Histone macroH2A has a novel hybrid structure consisting of a large nonhistone region and a region that closely resembles a full-length histone H2A. One key to understanding macroH2A function is determining the structure and function of its nonhistone region. The nonhistone region of one of the two known macroH2A subtypes was expressed in Escherichia coli and purified using affinity and molecular sieve chromatography. Crystals of the protein suitable for structural studies were grown from polyethylene glycol solutions by vapor equilibration techniques. The crystals belong to the hexagonal space group P6(4) (or its enantiomorph P6(2)) with unit cell parameters: a = b = 106.2 A, c = 125.9 A, alpha = beta = 90 degrees, and gamma = 120 degrees. There are four molecules in the asymmetric unit. Self-rotation function studies revealed three twofold noncrystallographic rotation axes related approximately by 222 symmetry. These crystals have 47% solvent content and diffract to 3.8 A resolution.

Leucine zipper motif in porcine renin-binding protein (RnBP) and its relationship to the formation of an RnBP-renin heterodimer and an RnBP homodimer.

An apparent leucine zipper motif was recognized in the predicted amino acid sequence of porcine kidney renin-binding protein (RnBP) by analysis of the nucleotide sequence of a cDNA encoding the protein (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem. 265, 6556-6561). To evaluate the role of this motif in the formation of an RnBP-renin heterodimer and an RnBP homodimer, a porcine mutant cDNA involving Leu185----Asp and Leu192----Asp substitutions was constructed and expressed in vitro and in Xenopus oocytes. The mutant protein neither binds to renin nor forms the homodimer. The results strongly suggest that the leucine zipper motif in the RnBP molecule mediates the formation of both the RnBP-renin heterodimer and the RnBP homodimer observed previously. The existence of the motif should facilitate elucidation of the role of RnBP in renin metabolism.

Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.