In this context, computational Selleckchem Epacadostat approaches for protein 3D modeling may assist in establishing genotype–phenotype and structure–function correlations, as well as predicting the structural and/or functional impact of each mutation. The goal of the present study was to identify the mutation(s) associated with odonto-HPP affecting monozygotic twin probands, establish a genotype–phenotype association, and use a 3D modeling approach to evaluate the impact of each mutation in the TNAP protein structure. Additionally, we evaluated the

expression of mutant protein and its subcellular localization in dental pulp cells from probands by Western blotting and immunocytochemistry. The probands were male monozygotic twins of Caucasian descent clinically diagnosed with odonto-HPP. Probands and their biological parents were examined in order

to identify potential mutations in the ALPL gene. The family was provided with study information and consented to participate (IRB #065/2005). The clinical diagnosis of odonto-HPP in the probands (by physical and dental examinations, radiographs, and blood chemistry assays) and subsequent Hydroxychloroquine datasheet management of dental symptoms have been reported previously [18] and [19]. Briefly, probands (patients A and B), at the age of two, were brought to the Piracicaba Dental School, University of Campinas, Brazil for dental evaluation. Parents reported premature exfoliation of the anterior primary teeth, with signs of partial root resorption. Physical examination and radiographs (long bones, joints, and skull) showed age-appropriate growth and development. Routine laboratory testing revealed low serum ALP activity for both probands (patient A: 62 U/L, patient B: 63 U/L; normal range for children 151–471 U/L), while serum phosphate and calcium levels remained within normal limits [18], [19] and [20]. Genomic DNA of probands and their parents

was isolated from peripheral blood leukocytes using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) Demeclocycline following the manufacturer’s instructions. Primer sequences were designed to amplify all TNAP coding exons (2–12), as previously reported [21], allowing analysis of the whole coding sequence, including intron–exon borders. Polymerase chain reaction (PCR) was performed in a final volume of 50 μL with 100 ng of DNA, 30 μM forward and reverse primers, 0.2 mM dNTP mix (Invitrogen™, Life Technologies, Carlsbad, CA, USA Life Technologies, Gaithersburg, MD, USA), 0.75 U Gold Tap® Flexi DNA polymerase (Promega), and 1–3 mM MgCl2. Cycle conditions and annealing temperature were optimized for each primer pair.