- Email: [email protected]
198-P HLA genes as markers in some rheumatological diseases
S140
Abstracts
197-P
POLYMORPHISM Ser326Cys OF GEN hOGG1 IN PROSTATE CANCER PATIENTS. Javier Garcia,1 Alberto J. Leon,2 Victoriano J. Leon,3 Maniuel Urrutia.1 1Servicio de Urologia, Hospital Universitario de Salamanca, Salamanca, Spain; 2Division of Experimental Therapeutics, Toronto General Research Institute, Toronto, ON, Canada; 3Servicio de Analisis Clinicos, Hospital Universitario de Salamanca, Salamanca, Spain. Aim: The accumulation of damages in the DNA of the prostate cells along the time, seems to play an important paper in the pathogenesis of prostate cancer patients, PC. DNA damage accumulates especially in mtDNA over time, and hOGG1 is an enzyme responsible for the repair of DNA oxidative damage either in nuclei and mitochondria. The Ser326Cys polymorphism was shown to diminish the hOGG1 activity in vitro, and it was suggested that the 326Cys allele might pose a higher risk of 8-OHdG formation in DNA. Recently, some authors have demonstrated that the capacity to repair oxidative DNA damage was significantly decreased in healthy human subjects bearing the hOGG1 Cys326Cys homozygous variant genotype, compared to wild type individuals. In the present , we intend to study the possible relation of this polymorphism with PC. Methods: Prostatic tissue from 16 cases of PC, and 94 controls, from peripheral blood lymphocytes; genomic DNA was extracted by means of the DNA direct II (Dynal) kit. Genotyping for the hOGG1 Ser326Cys polymorphism was performed by PCR-RFLP technique, briefly, a 234 bp product was amplified using the forward: 5’-CCC AAC CCC AGT GGA TTC TCA TTG C-3’ and the reverse: 5’-GGT GCC CCA TCT AGC CTT GCG GCC CTT-3’ primers. PCR conditions were 4 min at 94 °C and followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. Overnight digestion at 37 °C with Fnu4HI resulted in 213- and 21-bp products for the wild type allele (Ser326), and in 164-, 49- and 21-bp products for the mutant allele (Cys326). Digestion products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide. Results: The distribution of polymorphism in PC was: 62,3% SS, 33% SC 4,7 % CC, in the control group: 66,4 SS, 30,4 SC 3,2 CC. Conclusions: The Chi-square test, p⬍ 0.01. There are not correlation in polymorphism in this study inter PC and control group.
198-P
HLA GENES AS MARKERS IN SOME RHEUMATOLOGICAL DISEASES. Alberto J. Leon,1 Victoriano J. Leon,2 Susana Gomez Castro,3 Maria Dolores Sanchez Gonzalez.3 1Division Experimental Therapeutics, Toronto General Research Institute, Toronto, ON, Canada; 2Servicio Analisis Clinicos, Hospital Universitario de Salamanca, Salamanca, Spain; 3Servicio de Reumatologia, Hospital Universitario de Salamanca, Salamanca, Spain. Aim: Epidemiological studies of HLA alleles related with susceptibility or resistance to certain diseases present the problem of the high price of classical genomic HLA studies. In order to select those patients of interest that should be included in the HLA genomic studies, we have analized HLA A3, B7, DR2 and DR4 allele by PCR. Methods: 96 patients with different rheumatological diseases (50Rheumatoid Arthritis, RA; 46 , Psoriatic Arthritis PA) and 124 healthy controls were included in our study. DNA was stracted with the DNA Direct II kit (Dynal), which uses resin balls with a magnetic core in order to isolate DNA. The genes were analyzed by PCR using the following primers: HLA A3 : For 5’ AgC gAC gCC gCg AgC CA 3’ , Rev 5’ CAC TCC Acg CAC gTg CCA 3’ ; para HLA B7: For 5’ ggA gTA TTg ggA CCg gAA C 3’, Rev 5= TAC Cag CgC gCT CCA gCT 3’; para DR2 : For 5= TCC TgT ggC AgC CTA AgA g 3=, Rev 5= CgC TgC ACT gTg AAg CTC TC 3= y DR4: For 5=gTT TCT Tgg AgC Agg TTA AAC A 3=, Rev 5= CgC TgC ACT gTg AAG CTC TC 3=. PCR condicions were as follows: 95°, 5 min, 1 cycle; 95° 30 sec, 53° 30 sec, 72° 30sec 34 cycles, and 72° 10 min. PCR products were visualized by electrophoresis with agarose 2%. Results: Allelic frequencies for HLA A3 was 21,3 %, in RA, 19,2 % PE (13,6% en CG); in HLA B7 18,3 % RA, 17,5 % PA ( 12,5 % in CG); DR 2 17,6 % in RA, 17,2 PA (15,5 % in CG); DR4 32,4% RA, 28,3% PA ( 15,6% CG). Conclusions: They were similar to those previously published, significant differences were found between controls and RA or PA (The Chi-square test, p⬍ 0.001, between patients and control group).
Abstracts
197-P
POLYMORPHISM Ser326Cys OF GEN hOGG1 IN PROSTATE CANCER PATIENTS. Javier Garcia,1 Alberto J. Leon,2 Victoriano J. Leon,3 Maniuel Urrutia.1 1Servicio de Urologia, Hospital Universitario de Salamanca, Salamanca, Spain; 2Division of Experimental Therapeutics, Toronto General Research Institute, Toronto, ON, Canada; 3Servicio de Analisis Clinicos, Hospital Universitario de Salamanca, Salamanca, Spain. Aim: The accumulation of damages in the DNA of the prostate cells along the time, seems to play an important paper in the pathogenesis of prostate cancer patients, PC. DNA damage accumulates especially in mtDNA over time, and hOGG1 is an enzyme responsible for the repair of DNA oxidative damage either in nuclei and mitochondria. The Ser326Cys polymorphism was shown to diminish the hOGG1 activity in vitro, and it was suggested that the 326Cys allele might pose a higher risk of 8-OHdG formation in DNA. Recently, some authors have demonstrated that the capacity to repair oxidative DNA damage was significantly decreased in healthy human subjects bearing the hOGG1 Cys326Cys homozygous variant genotype, compared to wild type individuals. In the present , we intend to study the possible relation of this polymorphism with PC. Methods: Prostatic tissue from 16 cases of PC, and 94 controls, from peripheral blood lymphocytes; genomic DNA was extracted by means of the DNA direct II (Dynal) kit. Genotyping for the hOGG1 Ser326Cys polymorphism was performed by PCR-RFLP technique, briefly, a 234 bp product was amplified using the forward: 5’-CCC AAC CCC AGT GGA TTC TCA TTG C-3’ and the reverse: 5’-GGT GCC CCA TCT AGC CTT GCG GCC CTT-3’ primers. PCR conditions were 4 min at 94 °C and followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. Overnight digestion at 37 °C with Fnu4HI resulted in 213- and 21-bp products for the wild type allele (Ser326), and in 164-, 49- and 21-bp products for the mutant allele (Cys326). Digestion products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide. Results: The distribution of polymorphism in PC was: 62,3% SS, 33% SC 4,7 % CC, in the control group: 66,4 SS, 30,4 SC 3,2 CC. Conclusions: The Chi-square test, p⬍ 0.01. There are not correlation in polymorphism in this study inter PC and control group.
198-P
HLA GENES AS MARKERS IN SOME RHEUMATOLOGICAL DISEASES. Alberto J. Leon,1 Victoriano J. Leon,2 Susana Gomez Castro,3 Maria Dolores Sanchez Gonzalez.3 1Division Experimental Therapeutics, Toronto General Research Institute, Toronto, ON, Canada; 2Servicio Analisis Clinicos, Hospital Universitario de Salamanca, Salamanca, Spain; 3Servicio de Reumatologia, Hospital Universitario de Salamanca, Salamanca, Spain. Aim: Epidemiological studies of HLA alleles related with susceptibility or resistance to certain diseases present the problem of the high price of classical genomic HLA studies. In order to select those patients of interest that should be included in the HLA genomic studies, we have analized HLA A3, B7, DR2 and DR4 allele by PCR. Methods: 96 patients with different rheumatological diseases (50Rheumatoid Arthritis, RA; 46 , Psoriatic Arthritis PA) and 124 healthy controls were included in our study. DNA was stracted with the DNA Direct II kit (Dynal), which uses resin balls with a magnetic core in order to isolate DNA. The genes were analyzed by PCR using the following primers: HLA A3 : For 5’ AgC gAC gCC gCg AgC CA 3’ , Rev 5’ CAC TCC Acg CAC gTg CCA 3’ ; para HLA B7: For 5’ ggA gTA TTg ggA CCg gAA C 3’, Rev 5= TAC Cag CgC gCT CCA gCT 3’; para DR2 : For 5= TCC TgT ggC AgC CTA AgA g 3=, Rev 5= CgC TgC ACT gTg AAg CTC TC 3= y DR4: For 5=gTT TCT Tgg AgC Agg TTA AAC A 3=, Rev 5= CgC TgC ACT gTg AAG CTC TC 3=. PCR condicions were as follows: 95°, 5 min, 1 cycle; 95° 30 sec, 53° 30 sec, 72° 30sec 34 cycles, and 72° 10 min. PCR products were visualized by electrophoresis with agarose 2%. Results: Allelic frequencies for HLA A3 was 21,3 %, in RA, 19,2 % PE (13,6% en CG); in HLA B7 18,3 % RA, 17,5 % PA ( 12,5 % in CG); DR 2 17,6 % in RA, 17,2 PA (15,5 % in CG); DR4 32,4% RA, 28,3% PA ( 15,6% CG). Conclusions: They were similar to those previously published, significant differences were found between controls and RA or PA (The Chi-square test, p⬍ 0.001, between patients and control group).