Appropriate DNA fragments of leptin gene -18G > A, leptin receptor gene K109R and Q223R were amplified using PCR and analyzed using PCR-RFLP (Restriction Fragments Length Polymorphism), DHPLC (Denaturing High Performance Liquid Chromatography) or direct sequencing. The primer sequences are shown in table 2. Table 2 Sequences of primers Genetic polymorphism Sequences of primers Genotyping method used (restriction enzyme) Leptin gene – 18G > A tggagccccgtaggaatcgca tgggtctgacagtctcccaggga PCR-RFLP (AciI) Leptin receptor gene

– K109R tttccactgttgctttcgga aaactaaagaatttactgttgaaacaaatggc PCR-RFLP (HaeIII) Leptin receptor gene – Q223R aaactcaacgacactctcctt tgaactgacattagaggtgac PCR-RFLP (MspI) Statistical analysis The correlations of the genetic polymorphisms, biochemical test results, and overweight status were analyzed with regard to gender, intensity of chemotherapy (high intensity vs. standard intensity regimens) and to the use of CRT. Results #GS-4997 manufacturer randurls[1|1|,|CHEM1|]# were expressed as mean ± SEM. The data were analyzed by ANOVA followed by Scheffe’s post hoc test. For between-group comparison of nonparametric variables Chi2 test was used. Correlations between the variables were calculated using Pearson correlation. The P values < 0.05 were considered statistically significant.

MI-503 price The statistical analyses were performed using the Statistica 8 software package (Stat Soft, Inc., USA). Permanent Ethical Committee for Clinical Studies of the Medical College of the Jagiellonian University approved the study protocol. All parents, adolescent patients and adult patients signed written informed consent before blood sample collection. No patient refused participation in the study. Results Anthropometric evaluation Median BMI percentiles at the time HAS1 of ALL diagnosis and at the time of the study were 45.3 (m:0; M:99.6) and 65.5 (m:0.3; M:99.6), respectively. After the completion of ALL treatment BMI ≤ 10 percentile and ≥ 95 percentile was found in 9% and 13% of patients, respectively. At ALL diagnosis 21% of patients were classified as overweight (BMI ≥ 85), the respective proportion

at the time of the present study was 31%. The prevalence of the overweight status at the time of ALL diagnosis/after ALL treatment in patients treated with and without CRT was 10%/23% and 20%/35%, respectively (table 3). Table 3 Anthropometric evaluation Patients Total CRT No CRT   Number of patients (%) Total 82 (100) 31 (38) 51 (62) Gender:       Female 37 (45) 16 (20) 21 (26) Male 45 (55) 15 (18) 30 (36) Overweight at ALL diagnosis 13 (16) 3 (10) 10 (20) Overweight after ALL treatment 25 (31) 7 (23) 18 (35) CRT – cranial radiotherapy Leptin and soluble leptin receptor Significant differences were found between leptin levels in patients treated with and without CRT (figure 1) both in the entire study population (22.2+/- 3.13 ng/ml vs. 14.9+/-1.6 ng/ml; p < 0.03) and in female patients (29.9+/-4.86ng/ml vs. 16.9+/-2.44 ng/ml; p = 0.014).