Molecular modelling of novel ADCY3 variant predicts a molecular target for tackling obesity

The present study aimed to identify ADCY3 genetic variants in severely obese young patients of Greek-Cypriot origin by genomic sequencing. Total genomic DNA samples were isolated from peripheral whole blood using the Gentra Puregene Blood kit (Qiagen GmbH). DNA sequencing was performed with 100 ng genomic DNA, which was amplified using primers designed by Primer3 software ver. 0.4.0 (http://frodo.wi.mit.edu/). PCR mixtures were prepared using the Taq DNA Polymerase Kit (Qiagen GmbH); they had a final volume of 20 μl and contained 2 μl PCR buffer (10X), 2 μl Q Solution (5X), 2 μl dNTPs (2mM), 0.3 μl of each primer (10μM), 0.2 μl Taq polymerase (5U/μl) and 100 ng genomic DNA. Amplification was performed with an initial denaturing temperature at 95˚C for 5 min, followed by 30 cycles of denaturation (95˚C, 45 sec), annealing (57˚C, 60 sec) and extension (72˚C, 45 sec), with a final extension at 72˚C for 5 min. The ADCY3 gene primers covered all exons. The PCR products were analysed on an Applied Biosystems 3130xl Genetic Analyzer and the results were analysed using Sequencing Analysis R 5.3 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). The results revealed a total of five variants in patients, four of which were previously reported. A novel variant was identified in two patients (6%). The novel variant involves a heterozygous c.349T>A change in exon 1 of the gene locus, leading to a missense p.Leu117Met substitution.