G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.
Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318_1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggest a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the Probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization.
The assembly of a phenotype into modules or developmental fields, which are semiautonomous units in development and function, seems to be one of the strategies to increase the capacity to produce phenotypic variation. In mammals the upper dentition is formed on two distinct developmental units, wherein incisors are formed on the primary palate, which is derived from the embryonic frontonasal process, and the other teeth (canine, premolar, and molar) are formed on the alveolar bone, which is derived from the maxillary process (termed herein as PALATE2). The aim of the present work was to analyze the variations in size and number of premolar and molar teeth in primate dentition and to correlate these morphometrical parameters with the relative size of these tooth classes with respect to PALATE2. Furthermore, we seek to understand to what extent the changes in the relative size of premolar and molar fields can influence the size of each tooth within its respective field, and how these parameters connect with the variations in the dental formula that occurred during primate evolution. The data presented here not only indicate that premolar and molar fields can be seen as submodules of a larger and hierarchically superior module (i.e., PALATE2) but also present quantitative parameters that allow us to understand how variations in the relative size of premolar and molar teeth connect with the variations in the dental formula that occurred during primate evolution. Anat Rec,
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