Determination of the RhC/c Blood Group by Polymerase Chain Reaction with Sequence-specific Primers of the Intron 2 Insert of the RHCE Gene / 대한수혈학회지

BACKGROUND: RhC/c blood group antigens are of clinical importance and molecular genotyping for them can be useful when serological typing is difficult. A method to determine the RhC/c genotype, by targeting exon 1 nt48 and exon 2 nt307, has been used. However, this approach is not accurate for the RHc(cyt48) variant allele. We applied a more accurate genotyping method, using the intron 2 109 bp insert of the RHCE gene, and evaluated its performance in comparison with the standard method. METHODS: RhD and RhC/c serotypes of 236 subjects were determined. We compared two genotype results with the serological phenotype. One method examined the allele-specific exon 1 nt48 and exon 2 nt307 polymorphism area (Method 1), while the other method detected the intron 2 insert instead of the exon 1 nt48 (Method 2) by polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The predicted phenotypes by Method 1 were not matched with the true phenotypes in 24 cases (24/236, 10.2%). By contrast, the predicted results by Method 2 matched with true phenotypes in all cases except one. The RHc(cyt48) variant was suspected in 22 cases (23.7%) of the 93 Rhc cases. CONCLUSION: For the determination of the RhC/c genotype in Koreans, the method that analyzes exon 1 nt48 is inaccurate. Instead, intron 2 insert analysis with exon 2 nt307 by PCR-SSP appears to be a more accurate alternative.

Genetic Polymorphisms of UGT1A and their Association with Clinical Factors in Healthy Koreans

Glucuronidation by the uridine diphosphateglucuronosyltransferase 1A enzymes (UGT1As) is a major pathway for elimination of particular drugs and endogenous substances, such as bilirubin. We examined the relation of eight single nucleotide polymorphisms (SNPs) and haplotypes of the UGT1A gene with their clinical factors. For association analysis, we genotyped the variants by direct sequencing analysis and polymerase chain reaction (PCR) in 218 healthy Koreans. The frequency of UGT1A1 polymorphisms, -3279T>G, -3156G>A, -53 (TA)(6>7), 211G>A, and 686C>A, was 0.26, 0.12, 0.08, 0.15, and 0.01, respectively. The frequency of -118 (T)9>10 of UGT1A9 was 0.62, which was significantly higher than that in Caucasians (0.39). Neither the -2152C>T nor the -275T>A polymorphism was observed in Koreans or other Asians in comparison with Caucasians. The -3156G>A and -53 (TA)6>7 polymorphisms of UGT1A were significantly associated with platelet count and total bilirubin level (p=0.01, p=0.01, respectively). Additionally, total bilirubin level was positively correlated with occurrence of the UGT1A9-118 (T)(9>10) rare variant. Common haplotypes encompassing six UGT1A polymorphisms were significantly associated with total bilirubin level (p=0.01). Taken together, we suggest that determination of the UGT1A1 and UGT1A9 genotypes is clinically useful for predicting the efficacy and serious toxicities of particular drugs requiring glucuronidation.

Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Enterovirus is a common cause of aseptic meningitis, respiratory disease and nonspecific febrile illness. The conventional methods for laboratory diagnosis of enterovirus infections have been virus culture and serotyping by an immunofluorecent test. We studied a new and more rapid approach for enterovirus detection in cerebrospinal fluid (CSF) by real-time nested PCR. METHODS: This study was performed on 50 CSF specimens from patients suspected of aseptic meningitis. Enterovirus was detected in CSF by PCRs for 3 different targets and real-time nested PCR. Enterovirus culture was also performed in 44 CSF specimens. RESULTS: The positive rate of PCRs for each of the 3 different targets was 26.0%, 40.0%, or 46.0%, and that of real-time nested PCR was 86.0%. Only 6.8% were positive in culture. Thus, the positive rate of real-time nested PCR was much higher than other methods. CONCLUSIONS: Our study revealed that the real-time nested PCR should be useful for diagnosis of enterovirus infections because of a high sensitivity and rapid detection.

Genotype and allele frequencies of the platelet glycoprotein genes associated with arterial thrombosis in Korean population / 대한수혈학회지

BACKGROUND: Ischemic heart disease and cerebrovascular disease are the main causes of death and platelets are responsible for the formation of arterial thrombi. Platelet membrane glycoproteins (GP) associated with coagulation pathway are GPIb/V/IX, GPIa/IIa, and GPIIb/IIIa. We evaluated genotype and allele frequencies of seven platelet glycoprotein genes associated with arterial thrombosis. METHODS: Genomic DNA was isolated from peripheral blood of 300 unrelated Korean and single nucleotide polymorphism of platelet glycoproteins was analyzed. PCR with sequence specific primers was used to investigate GPIa C807T and GPIbalpha VNTR polymorphism. PCR-RFLP (restriction fragment length polymorphism) was used to investigate GPIa G1648A and C2531T, GPIbalpha C524T and T-5C, and GPIIIa T1565C polymorphism. RESULTS: The allele frequencies of GPIa C807T were 807C 0.733, 807T 0.267; GPIa 1648G 0.975, 1648A 0.025; GPIa C2531T, 2531C 1.000, 2531T 0.000; GPIbalpha C524T, 524C 0.927, 524T 0.073; GPIbalpha VNTR, A 0.017, B 0.015, C 0.558, D 0.410; GPIbalpha T-5C, -5T 0.726, -5C 0.274; GPIIIa T1565C, 1565T 0.995, 1565C 0.005. CONCLUSION: The genotype and allele frequencies of GPIa G1648A, GPIbalpha C524T, and GPIIIa T1565C were similar to established data. GPIa 807T and -5T allele of Kozak polymorphism showed low frequency compared with other ethnic group. Allele frequencies of GPIbalpha VNTR A and B alleles were very alike (0.017 vs 0.015). In this study, we firstly evaluated the genotype and allele frequencies of GPIa C2531T and GPIbalpha VNTR, T-5C polymorphisms in Korean population. This study will serve as a basic data for the study of platelet glycoproteins associated with arterial thrombosis in Korean.

Detection of Helicobacter pylori and BabA (Blood-group Antigen Binding Adhesin) in Saliva and Gastric Tissue by Polymerase Chain Reaction / 임상검사와정도관리

BACKGROUND: Saliva is considered an important vector for the Helicobacter pylori infection. The presence of the babA2 gene, encoding for BabA (blood-group antigen binding adhesin), in the H. pylori genome is crucial for H. pylori-related pathogenesis. METHODS: The study was performed in the group of 215 patients. The detection of H. pylori and babA2 in saliva and gastric tissue was done by PCR (polymerase chain reaction). Moreover, gastric tissues were stained with hematoxylin-eosin as well as with modified Giemsa methods for the analysis of Helicobacter pylori density. RESULTS: The positive rate of H. pylori by nested PCR was 78.6% in gastric tissue and 72.7% in saliva. In addition, the positive rate of H. pylori was 55.5% by the histological analysis of Helicobacter pylori density in gastric tissue. The positive rate of babA2 by PCR was 33.9% in gastric tissue, and 8.2% in saliva. CONCLUSION: We revealed that the H. pylori PCR results obtained in gastric tissue correlated well with those obtained in saliva. As saliva is more available specimen, it is more suitable for clinical application of H. pylori detection by PCR. However, clinical use of - BabA PCR seems to be limited because of its low-sensitivity.