Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

The development of a prophylactic vaccine that targets human papillomaviruses (HPV) 6, 11, 16, and 18 to prevent cervical cancer has increased interest in the ethnic and geographical distributions of HPV genotypes. We investigated HPV prevalence and type distribution by restriction fragment mass polymorphism (RFMP) testing a total of 60,775 specimens (aged 18-79 yr, median 44) taken from liquid-based cytology. Overall HPV positive rate of total patients was 34.2%. Among the positive patients, 87.7% was single type infections, and 12.3% was multiple HPV types. HPV-16 was the most prevalent genotype observed in 2,307 (26.0%), followed by type 52 in 2,269 (25.5%), type 58 in 1,090 (12.3%), type 18 in 633 (7.1%), type 56 in 436 (4.9%). The pattern of high risk-HPV positive rate according to age showed U-shape with a peak in HPV prevalence among women less than 30 yr of age, and a second peak among the older females aged 70 to 79 yr. The leading four high-risk HPV genotypes were HPV-16, HPV-52, HPV-58, and HPV-18 in descending order. In conclusion, this study provides the most representative prevalence and type-specific distribution of HPV among Korean women, and demonstrates that the epidemiology of HPV infection is different from that of other regions of the world.

Human Papilloma Virus Genotyping Assay using Restriction Fragment Mass Polymorphism Analysis, and Its Comparison with Sequencing and Hybrid Capture Assays / 대한진단검사의학회지

BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.

Single Nucleotide Polymorphisms in the 3'-Untranslated Region of Vascular Endothelial Growth Factor in Diabetic Retinopathy

PURPOSE: Recent studies suggest that increased expression of the vascular endothelial growth factor (VEGF) may play a role in the pathogenesis of diabetic complications. The aim of this study was to assess the potential association of VEGF gene polymorphisms with disease expression of retinopathy in patients with diabetes mellitus. METHODS: We studied 96 diabetic patients to determine whether there is an association between diabetic retinopathy in VEGF and VEGFR gene; all patients were unrelated Korean individuals (PDR, n=32; NPDR, n=25; no DMR, n=39). We analyzed VEGF and VEGFR SNP by restriction fragment mass polymorphism (RFMP). PCR was performed with primers designed to introduce a type IIS restriction endonuclease recognition sequence (FokI, BstF5I) ahead of the polymorphism site. The enzymatic cleavage of the products resulted to excision of two oligonucleotide fragments containing the variation site, and then masses of the resulting oligonucleotide fragments were examined by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS). Difference are seen as the presence, absence, or mass change in peaks corresponding to the fragments affected by existence of polymorphism that have base substitution at the site of variation. RESULTS: We found that the genotype frequencies of two polymorphisms shown to be completely linked to each other (rs3025039; vf1, rs3025040; vf2) and one polymorphism (rs3812867; vfr1) within 3'-untranslated regions (3'-UTR) of the VEGF and VEGFR1 significantly differed between patients with and without retinopathy. The frequencies of the vf1, vf2 and vfr1 did not differ significantly between the NPDR and PDR group CONCLUSIONS: These data implicate the polymorphisms in the 3'-UTR of the VEGF and VEGFR1 genes as risk factors for diabetic retinopathy.

The Comparison of Restriction Fragment Mass Polymorphism with Sequencing Method for the Hepatitis C Virus Genotyping / 대한진단검사의학회지

BACKGROUND: Hepatitis C virus (HCV) genotyping is of increasing the importance in the progression to liver cirrhosis and the outcome of antiviral therapy. Restriction fragment mass polymorphism (RFMP) method which is developed recently for HCV genotyping is known to report accurate result in mixed infection. We evaluated the performance of RFMP in HCV genotyping by comparing the result of direct sequencing. METHODS: Forty-three chronically HCV infected patients in Asan Medical Center were enrolled. HCV genotyping was performed by both RFMP and direct sequencing. The results were compared from the genotype level to the subtype level. RESULTS: In the genotype level, all the results (100%) were concordant. At the subtype level, however, 2 results (2/43, 4.7%) were disconcordant. Two cases were reported as single infection of type 1b by direct sequencing while RFMP reported as mixed infection of 1b with 1a and 1b with 1c, respectively. CONCLUSIONS: Both sequencing and RFMP assays were highly concordant. We suggest that RFMP is a novel method in HCV genotyping superior to sequencing or hybridization method especially in mixed infection.

The Comparison of Restriction Fragment Mass Polymorphism Method with Sequecing Method for the Detection of HBV YMDD Mutants / 대한진단검사의학회지

BACKGROUND: YMDD motif mutants of the hepatitis B virus (HBV) emerged in some chronic hepatitis B patients after prolonged lamivudine treatment. Recently a novel genotyping assay, the restriction fragment mass polymorphism (RFMP) method, was introduced for the detection of YMDD mutations. We compared the performance of the RFMP method with that of sequencing method in chronic hepatitis B patients who had suffered the HBV DNA breakthrough after lamivudine treatment. METHODS: Enrolled in this study were 18 chronic hepatitis B patients who experienced the DNA breakthrough after a period during which HBV DNA was undetectable by Hybrid capture II HBV DNA test (Digene Inc., Gaithersburg, MD, USA). Both sequencing and RFMP methods were used to detect YMDD variants in three phases such as before treatment, before breakthrough and after breakthrough. RESULTS: YMDD mutants were detected in 13 samples (72.2%) by both methods after DNA breakthrough. Among them were two samples with a mixed HBV population detected by RFMP. Before breakthrough, the mutants were detected in three samples (16.7%) by sequencing and four (22.2%) by RFMP, showing discrepant results for two samples. The concordance rate between both methods was 92.6%. CONCLUSIONS: Both sequencing and RFMP methods were highly concordant except in a few cases, so it is suggested that both methods are appropriate in detecting YMDD mutants.

Reappraisal of HBV Genotypes and Clinical Significance in Koreans Using MALDI-TOF Mass Spectrometry / 대한간학회지

BACKGROUND/AIMS: Recent studies have shown that the genotype of hepatitis B virus (HBV) may correlate with the disease natural history and treatment outcome. However, several of these studies used low sensitivity assays in a small number of patients, and this has precluded an accurate evaluation of Korean HBV genotypes. We analyzed Korean HBV genotypes in a large population by employing a new technology, restriction fragment mass polymorphism (RFMP) using MALDI-TOF mass spectrometry, in a sensitive and specific manner. METHODS: Between February 1995 and December 2003, a total of 475 patients with chronic HBV infection were enrolled. The assay is based on the mass measurement of oligonucleotides having genotypic variations of the S gene. Clinical features including the virologic status and disease progression were also evaluated. RESULTS: The median age of the total patients was 35.5 years. Out of 475 patients, there were 162 (34.1%) inactive carriers, 172 (36.2%) had chronic hepatitis, 77 (16.2%) had liver cirrhosis and 64 (13.5%) had hepatocellular carcinoma (HCC). There were 454 patients (95.6%) with genotype C, 4 patients (0.8%) with genotype A, 16 patients (3.4%) with the mixed A and C genotype [7 patients (1.4%) with AA], and 1 patient (0.2%) with B genotype. Comparing genotype A and C, genotype A patients were all inactive carriers without HCC, whereas genotype C patients included those with chronic hepatitis, liver cirrhosis and HCC. CONCLUSIONS: HBV genotype C is highly prevalent in Korea. Although it is a small percentage, genotype A also exists and it seems to take a more benign clinical course than genotype C. Further studies are necessitated to assess the relationship between HBV genotypes and the various aspects of the diseases' clinical course.