In all patients pharmacological treatment was prescribed when serum AP and BAP levels firstly increased at least 25% above the upper limit of the normal range. During the whole observation period we used in these PDB patients synthetic sCT at the dosage of 100 UI i.m/day for 6 months and bisphosphonates (clodronate, 300 mg i.v./day for 5 days, and neridronate, 100 mg/day i.v. for 2 days). As regards sCT, another course of treatment was prescribed after the sixth month of follow-up when total serum AP activity newly increased above the upper limit of the normal range by at least 25%. Overall, regarding bisphosphonates therapy, we have treated about 50 PDB outpatients. The nadir of our patients’ serum AP and BAP levels were observed between the third and the fifth month after therapy and the zenith usually after the ninth month (data not published). Disease activity was evaluated by the means of serum total AP and BAP determined at three, five, and nine months after therapy. Another course of drugs was prescribed when, after the ninth month, total serum AP and BAP had an increase of at least 25% above the upper limit of the normal range. After 5 months of treatment with clodronate, the patients showing serum AP and BAP above the upper limit of the normal range, although exhibiting a reduction of their values to 50% or more of the pre-treatment value, were classified as not responders to the therapy and switched to neridronate.
Genetic tests were performed in the affected patients VB and VG, and in VS, not affected. Genetic tests of their father were not possible, because the study started ten years after his death and no biological samples were available. After administration of an informed consent form approved by the Local Ethical Committee, peripheral blood was obtained and genomic DNA was extracted using a microvolume extraction method, QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. PCR protocol was performed as follows. Exons 7 and 8 of the SQSTM1/p62 gene were amplified by PCR (I-Cycler, Bio-Rad Laboratories, Milan, Italy) using two couples of primers located in the flanking introns: respectively 5′-GACTGTCT- GCCAGGAGCC-3’/5′-CCCTGCAGTGGAGAACATCT-3′ for exon 7 and 5′-CAGTGTGGCCTGTGAGGAC-3’/5′-CAGT- GAGCCTTGGGTCTCG-3′ for exon 8. For each patient we used 0.1 mg of DNA, in a final buffer volume of 50 ml [67 mM Tris-HCl, 16.6 mM (NH4)SO4, 0.01% Tween-20, 1.5 mM MgCl2, 0.2 mM deoxyribonucleotides, 0.2 mM of each primer and one unit of Polytaq (Polymed, Florence, Italy)]. Thirty-five PCR cycles were performed at 94 °C for 30 seconds, 58 °C (exon 7) or 55 °C (exon 8) for 30 seconds and 72 °C for 1 minute, after a first denaturing cycle at 94 °C for 3 minutes. A final extension cycle of 5 minutes at 72 °C was performed. PCR products were tested by 2% ethidium bromide-stained agarose gel electrophoresis, purified using the High Pure PCR. Can’t afford your medication? Buy cialis professional 20 mg
Product Purification Kit (Roche, Indianapolis, IN, USA) and finally sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction consisted of twenty-five repeated cycles of denaturation for 10 seconds at 96 °C, annealing for 5 seconds at 55 °C and extension for 2 minutes at 60 °C. The sequencing products were purified with DyeEx 2.0 Spin Kit (Quiagen, GmbH, Hilden, Germany) to remove the excess of dye terminator. Five microliters of each purified sequence were then resus- pended in 15 ml of formamide and denaturated for 2 minutes at 95 °C. Analysis of the forward and reverse sequences was performed on the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained were compared to reference sequence NM_003900.