Hypermethylation Analysis of p16INK4a and p15INK4b Promoters in Chronic Lymphocytic Leukaemia Patients and Normal Individuals

@#Introduction: Chronic Lymphocytic Leukaemia (CLL) is a common type of leukaemia in persons of predominantly European descent but is rare in the Asian population. Disparities in CLL incidence among people of Asian and European descent may be related to the genetic make-up of the two different populations. Hypermethylation event might be one of the silencing mechanisms that inactivate the tumour suppressor genes in CLL. The aim of this study was to determine the hypermethylation status of p16INK4a and p15INK4b among CLL patients and normal individuals. Materials & Methods: A total of 25 CLL patients and 25 normal individuals were recruited for this study and their genomic DNA were extracted from the peripheral blood. The hypermethylation status of p16INK4a and p15INK4b were determined using Methylation Specific-PCR (MS-PCR) whereas DNA sequencing method was applied to selected samples for validation of the MS-PCR results. We also evaluated the association between hypermethylation of these genes with the clinical and demographic characteristics of each group of subjects. Results: Among the CLL patients, p15INK4b partialmethylation occurred in 6 (24%) subjects while methylation occurred in 1 (4%) subject. All the remaining patients were unmethylated at p15INK4b. All the samples showed unmethylation at p16INK4a. Statistically significant associations were found between p15INK4b hypermethylation with the presence of CLL (p=0.01) and with race (p=0.02). Conclusion: Further study using a larger sample size is warranted to explore the significance of DNA methylation incidence among the CLL patients of the Malaysian population. Hence, we suggest that hypermethylation at p15INK4b has a huge influence that kick-starts CLL disease among Malaysians and MS-PCR technique is applicable to be used in methylation study.

Prevalence of Human Papillomavirus Genotypes in Routine Pap Smear of 2,562 Korean Women Determined by PCR-DNA Sequencing

The infections by human papillomaviruses (HPVs) are clearly associated with the subsequent development of cervical cancer. In this study, HPV genotype distribution and prevalence were detected in Korean women from January to December 2008 using PCR-DNA sequencing. A total of 2,562 cervical samples from Korean women having routine Pap smear cytology screening were used. HPV DNA was extracted from cervical swab samples and amplified by PCR in L1 region of HPV. HPV DNA was detected in 23.2% and 65.5% from the groups of normal and abnormal Pap cytology, respectively. The prevalence of high-risk types of HPV had the highest frequency in the <30 year-olds' group (50.6%). The prevalence of HPV in normal, ASCUS, LSIL and HSIL groups was 23.2%, 58.1%, 96.3% and 97.0%, respectively. Moreover, the frequencies of the high-risk types of HPV were 16.2% in the normal Pap cytology, 44.7% in the ASCUS, 76.1% in the LSIL and 94.1% in the HSIL groups. The prevalence of the high-risk types of HPV increased in proportion to the severity of the cytological classification. In the HSIL group, HPV type 16 was the most frequently found at 32.4%, followed by types 58, 53 and 33 at 17.6%, 14.7% and 11.8%, respectively. HPV type 82 was found in 5.6% of the HSIL group and was not detected in the normal Pap cytology group. The frequency of high-risk type of HPV 82 is firstly reported in Korean women. This finding could be an informative basis for the development of future HPV vaccination strategies in Korean women.

p53 Gene Mutations in Bladder Cancer (I): Comparative Analysis by Immunohistochemistry and DNA Sequencing / 대한비뇨기과학회지

Background. p53 gene mutations are known to play an important role in the progression of bladder cancer. Immunohistochemistry (IHC) has been used routinely to analyze p53 gene mutations by identifying nuclear overexpression of p53 protein. However, the accuracy and value of IHC as a marker of p53 gene mutation has been questioned. Methods. In this study of 35 bladder transitional cell carcinoma, we compared results of IHC staining with those of polymerase chain reaction (PCR) of exons 5 to 8 of p53 gene, followed subcloning of PCR products, and DNA sequencing analysis. Results. On IHC staining, 12 bladder tumors (37.5%) showed overexpression of p53 as defined by nuclear staining of 10% or more of tumor cells. On DNA sequencing analysis, 11 out of 32 cases (34.3%) showed point mutations in one or more exons of p53 gene. The results of IHC were concordant with that of DNA sequencing in 84.3% of cases. The sensitivity and specificity of detecting p53 mutations by IHC were estimated to be 81.8% and 90.5%, respectively. Conclusion. When properly used, IHC is a highly sensitive and specific, and clinically useful method to detect p53 gene mutations in bladder cancer.

A STUDY OF HUMAN MITOCHONDRIAL DNA POLYMORPHISM REGION SEQUENCING / 中国法医学杂志

By means of Polymerase chain reaction (PCR), the mitochondrial DNA polymorphous region from the base number 15997 to 16401 was amplified. After purification,the amplified fragment was directly Sequenced on automatic .DNA sequencer and accurate results was obtained. Compared with the former reports, there are base divergence between the results of this paper and sequence of Caucasian. Changes of base in six samples were observed.The numbers of base change are 3 to 6. The maternal inheritance of the mitochondrial genome makes mtDNA sequencing via PCR an attractive tool for testing whether individuals are maternally related. The method can also be used for studying of race inheritance and molecular evolution. The method is rather simpler and quicker than the former methods.