Análisis de mutaciones en los genes PINK1 Y PARKIN en pacientes colombianos con enfermedad de Parkinson / Analysis of mutations in the genes pink1 and parkin in Colombian patients with Parkinson's disease

La enfermedad de Parkinson es un desorden neurodegenerativo complejo, caracterizado por la pérdida progresiva de las neuronas dopaminérgicas de la sustancia nigra pars compacta. Factores tanto ambientales como genéticos se ha determinado que contribuyen a su desarrollo. Mutaciones en los genes PINK1 y PARKIN han sido asociadas con la enfermedad de inicio temprano e historia familiar. El objetivo del presente estudio fue identificar mutaciones en los genes PINK1 (exones 4 y 6) y PARKIN (exones 2 y 7) en 22 pacientes colombianos con EP de inicio temprano y/o antecedentes familiares, mediante amplificación por PCR y secuenciamiento. Las secuencias se compararon con la secuencia consenso de referencia. Se detectó una mutación homocigota de cambio en el marco de lectura ( frameshift) c.155 delA en el exón 2 del gen PARKIN en una paciente con inicio temprano de la enfermedad e historia familiar. Además se identificó la presencia de un polimorfismo en el intrón 2 del gen PARKIN en siete pacientes, uno de ellos en estado homocigoto. No se encontraron mutaciones en los exones 4 y 6 del gen PINK1. Se encontró una mutación homocigota c.155 delA en el exón 2 de PARKIN de una paciente con la enfermedad de Parkinson de inicio temprano con historia familiar. No se encontraron cambios el gen PINK1.

Parkinson's disease is a complex neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons of the substance nigra pars compacta. It has been determined that factors both environmental and genetic contribute to its development. Mutations in the genes PINK1 and PARKIN have been associated with the early onset of disease and family history. The goal of this study was to identify mutations in the PINK1 genes (exons 4 and 6) and PARKIN (exons 2 and 7) in 22 Colombian patients with EP of early onset and/or family history, by PCR amplification and sequencing. The sequences were compared with the reference consensus sequence. A homozygous change mutation was detected in the reading frame (frame shift) c.155 de la in exon 2 of the PAR-KIN gene in a patient with early onset of the disease and family history. In addition, the presence of a polymorphism in intron 2 of the PARKIN gene was identified in seven patients, one of them in homozygous state. Mutations were not found in exons 4 and 6 of the gene PINK1. A homozygous mutation c.155 de la in exon 2 of PARKIN was found in a female patient with Parkinson's disease early onset with family history. No changes to the gene PINK1 were found.

Application of Real-Time PCR for ABO Genotyping for Large-scale Population Screening / 대한수혈학회지

BACKGROUND: For large-scale population screening, the method of ABO genotyping needs to be simple, accurate and cost-effective. The real-time PCR method has been introduced and it is suitable for dealing with large numbers of specimens. In this study, we examined the ABO genotyping of 1,700 residents of Jeollanam-do for an epidemiologic study by applying the real-time PCR method. METHODS: Genomic DNA was extracted from the peripheral blood samples of 1,700 residents of Jeollanam-do between July 2004 and January 2006 and these samples were stored at -70degrees C. The ABO genotype in all the samples was determined by four-color real-time PCR using displacing probes and three cases that had an atypical real time PCR pattern were confirmed by direct sequencing and PCR-based cloning of exons 6&7 of the ABO gene. RESULTS: The genotyping results of 1,700 samples included O/O (25.6%), A/A (9.1%), A/O (29.1%), B/B (4.5%), B/O (19.8%) and A/B (11.9%), and the allele frequencies of O, A and B were 50.1%, 29.5% and 20.4%, respectively. The frequency of the O allele was lower in the residents of Jeollanam-do than that previously reported for the residents of Kangwon-do (P=0.014), while the frequency of the A allele was higher in the residents of Jeollanam-do than that previously reported for the residents of Kangwon-do (P=0.003). The three cases with atypical results were revealed to be B101/O24, Bvar(296C>T)/O01 and B101/Ovar(801G>T). It takes 6 days to perform ABO genotyping on 1,700 samples by a calculation per test. CONCLUSION: ABO genotyping by real-time PCR using displacing probes can be useful for mass screening for ABO genotyping. In Korea, the frequency of the ABO allele was significantly different among different regions.

Allele-related Variation in Minisatellite Repeats Involved in Transcription of the ABO Gene in Korean Blood Donors

BACKGROUND: The CBF/NF-Y enhancer region of ABO gene reported to contain 43bp minisatellite tandem repeats has been rarely reported. We describe here the relationship between minisatellite tandem repeats and ABO alleles in samples from Korean population with common ABO blood group and rare ABO subgroup. METHODS: Sixty one cases of ABO subgroup (14 A2, 12 A2B, 1 Aweak, 7 AweakB, 11 B3, 5 A1B3, 1 A1Bweak, 2 Bweak, and 8 cis-AB) and 41 cases of common ABO blood group (13 A, 6 AB, 11 B, and 11 O) were obtained from healthy donors at the Gwangju-Chonnam Red Cross Blood Center between Sep 2004 and Aug 2005. Red cells were phenotyped by standard serologic tests and genotyped by direct DNA sequencing exon 6 and 7 of the ABO gene. The minisatellite repeats were analyzed by PCR method. RESULTS: The ABO*A101 and *A102 had only one repeat, *B101, *O01 and *O02 had 4 repeats in common ABO blood group, while the *A102, *cis-AB01, and *Aw10 had only one minisatellite repeat and *A201, *A204, *B101, *Bw03, *B306, *O01, and *O02 alleles had 4 repeats and unexpectedly 3 A2 cases with *A102 had 4 repeats in the rare ABO subgroup. CONCLUSION: The minisatellite repeats found in Koreans correlate well with ABO alleles in sample common ABO phenotype, but do not completely correlate with those of ABO subgroup. We revealed here a pattern of the minisatellite repeats in various ABO subgroup in Korea.

p53 Gene Mutations in Astrtocytoma Detection by Direct DNA Sequencing / 대한암학회지

PURPOSE: Mutations in the p53 gene have been recognized in brain tumor, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for fresh frozen tissues. We have tried a method utilizing polymerase chain reaction (PCR) for the direct DNA sequecing of the p53 gene using a single 10 m paraffin-embedded tissue section. We applied this method to detect for p53 gene mutations in exons 5~8 in human astrocytoma utilizing paraffin-embedded tissues. MATERIALS AND METHODS: Twenty paraffin blocks containing tumor were selected from surgical specimens from twenty different cases. Tumors included 10 astrocytomas and 10 anaplastic astrocytomas. Ten controls were also selected among autopsy cases showing normal brain in light microscopy. The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure above 90% of the tumor cell population for p53 mutational analysis. RESULT: Mutation in the p53 gene was identified in 1 of 10 (10%) anaplastic astrocytomas. Mutations in the p53 gene were identified in 1 of 10 cases (10%) by PCR and direct DNA sequencing. Mutation in exon 7 resulting in amino acid substitution was found in one anaplastic astrocytoma (codon 245, GGC-->GAC: glycine-->aspartic acid). Ten control cases, ten astrocytomas and nine anaplastic astrocytomas were confirmed to be negative by direct sequencing of amplified DNA. CONCLUSION: This study demonstrates the feasibility of evaluating p53 gene mutations in archived astrocytoma specimens using PCR and direct DNA sequencing on paraffin sections. Application of this method should facilitate investigation of the role of p53 gene mutations in tumor biology.