The aim of this study was to investigate the feasibility of using RASSF1A gene as a universal fetal marker in maternal plasma. Two methods of circulating cell-free fetal DNA (cffDNA) extracted from maternal plasma were compared. The better one was chosen for extraction of cffDNA in the 20 pregnant samples. The SRY gene and the RASSF1A gene treated with methylation-sensitive restriction enzyme were amplificated by RT-PCR and the PCR system was optimized. The results showed that the SRY gene was found in 11 out of the 20 pregnant samples, which was consistent with the postnatal sex. Using the optimized PCR system, the specifically amplified fetal-associated methylated RASSF1A gene was found after treatment with BstUI in 18 of the 20 pregnant samples, while the 2 samples failed in detection. It is concluded that the methylated fetal-specific RASSF1A gene can be used as a universal fetal marker for the presence of cffDNA in maternal plasma without fetal gender restrictions. So, it can be used for noninvasive prenatal diagnosis.
DNA ; isolation & purification ; Female ; Fetus ; Humans ; Pregnancy ; Prenatal Diagnosis ; methods ; Tumor Suppressor Proteins ; blood ; genetics

The promyelocytic leukemia protein (PML), encoded by PML gene, plays a tumor suppressor in acute promyelocytic leukemia and other hematologic malignancies. Recent evidence indicates that PML involves in regulating multiple cell biological function, regulates self-renewal and maintains stable function in stem cell/cancer stem cell of multiple tissues, leading to drug resistance of cancer. This review summarizes the latest research advances about the relationship and therapeutic options between PML and cancer stem cell of hematologic neoplasms, aiming to propose a new avenue for blood cancer treatment.
Hematologic Neoplasms ; blood ; Humans ; Neoplastic Stem Cells ; Nuclear Proteins ; Promyelocytic Leukemia Protein ; Transcription Factors ; Tumor Suppressor Proteins

OBJECTIVETo detect hypermethylated tumor-specific RASSF1A DNA in the circulation and its significance in ovarian cancers patients.

METHODSMethylation-specific polymerase chain reaction (MSP) was used to study the hypermethylation of RASSF1A in preoperative serum samples from 51 ovarian cancer patients.

RESULTSThe RASSF1A gene was not methylated in peripheral blood samples from 51 normal patients and 51 patients with benign ovarian tumors. Hypermethylation of RASSF1A gene was found in circulating tumor-specific DNA in 43.1% of patients (22 out of 51 cases) with ovarian cancers (P < 0.05). There was no difference in hypermethylation of RASSF1A gene amongst various ovarian cancer subtypes (P < 0.05). On the other hand, hypermethylation of RASSF1A gene was more frequently encountered in stage III and IV than stage I and II tumors (P < 0.05). It was rarely seen in well and moderately differentiated groups, as compared with poorly differentiated group (P < 0.05).

CONCLUSIONSThere is a higher frequency of RASSF1A hypermethylation in circulating tumor-specific DNA of ovarian cancer patients. RASSF1A has been postulated to play an important role as tumor suppressor gene and can be silenced by promoter hypermethylation. This methylation correlates with clinical stage and histopathologic grade. Such observation may carry diagnostic and prognostic implications when assessing ovarian tumors.

Carcinoma, Endometrioid ; blood ; pathology ; Cystadenocarcinoma, Mucinous ; blood ; pathology ; Cystadenocarcinoma, Serous ; blood ; pathology ; DNA Methylation ; Female ; Humans ; Neoplasm Staging ; Ovarian Neoplasms ; blood ; pathology ; Tumor Suppressor Proteins ; blood ; genetics

Central precocious puberty (CPP) is caused by premature activation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin and G-protein coupled receptor-54 system is the essential gatekeeper of the reproductive system, playing a key role in the activation of the gonadotropic axis at puberty. We aimed to determine whether serum kisspeptin may function as a marker for CPP by investigating serum kisspeptin levels in Korean girls with CPP and their prepubertal controls. Serum kisspeptin levels of Korean girls with CPP (n = 30) and age-matched healthy prepubertal controls (n = 30) were measured with a competitive enzyme immunoassay. Serum kisspeptin levels were significantly higher in CPP group than in control group (4.61 +/- 1.78 vs 2.15 +/- 1.52 pM/L, P < 0.001). Serum kisspeptin was positively correlated with peak luteinizing hormone (LH), peak/basal LH ratio and peak LH/follicular-stimulating hormone (FSH) ratio during GnRH stimulation test. CPP is supposed to be triggered by premature increase of kisspeptin. Serum kisspeptin may be used as a marker of CPP. Further studies on KISS1 gene polymorphisms leading to higher risk of premature increase of kisspeptin and upstream regulator of kisspeptin are also needed.
Biological Markers/blood ; Child ; Female ; Follicle Stimulating Hormone/blood ; Humans ; Luteinizing Hormone/blood ; Puberty, Precocious/blood/*diagnosis ; Republic of Korea ; Tumor Suppressor Proteins/*blood

OBJECTIVETo investigate the mRNA expression and promoter methylation status of p73 gene in the peripheral blood of children with Wilms' tumor (WT), and their relationship.

METHODSForty-five children with WT were selected as the case group, and 15 sex- and age- matched children (without malignancies) who visited the hospital for physical examination or other reasons were selected as the control group. Peripheral blood was collected from both groups. Real-time quantitative PCR and methylation-specific PCR were used to determine the mRNA expression level and promoter methylation status of p73 gene. Their relationship with clinicopathological features and the effect of promoter methylation on mRNA expression of p73 gene were analyzed in the case group.

RESULTSThe relative quantity (RQ) of p73 mRNA in the case group was significantly higher than in the control group (3.2 ± 0.9 vs 1.6 ± 1.1; P<0.01). The positive rate of p73 gene promoter methylation in the case group was significantly lower than in the control group (20% vs 73%; P<0.01). In the case group, the RQ of p73 mRNA was significantly higher in children with methylated p73 gene promoter than in those with unmethylated p73 gene promoter (P<0.01). In children with methylated p73 gene promoter, the RQ of p73 mRNA was significantly higher in the case group than in the control group (P<0.01). In children with unmethylated p73 gene promoter, there was no significant difference in RQ of p73 mRNA between the case and control groups (P=0.810).

CONCLUSIONSAberrant promoter methylation of p73 gene in peripheral blood is one of the gene expression regulations in children with WT, and it is related to the onset and development of WT. The p73 gene may play a role as oncogene in WT patients with p73 gene promoter methylation and mRNA overexpression is associated with promoter methylation status of p73 gene.

Child, Preschool ; DNA Methylation ; DNA-Binding Proteins ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Infant ; Kidney Neoplasms ; genetics ; Male ; Nuclear Proteins ; genetics ; Promoter Regions, Genetic ; RNA, Messenger ; blood ; Tumor Protein p73 ; Tumor Suppressor Proteins ; genetics ; Wilms Tumor ; genetics

OBJECTIVETo investigate the feasibility of noninvasive fetal ABO genotyping based on RASSF1A gene with circulating cell-free fetal DNA(cffDNA) from maternal plasma.

METHODSDNA was extracted from the O group pregnant plasma, and the presence of cffDNA was confirmed by fetal DNA maker SRY and RASSF1A. B and non-O were detected by real-time PCR, and the genotyping results were evaluated by using the serologic tests for ABO phenotyping.

RESULTSAmong the samples of 20 cases, the SRY was found in 11 cases by detecteion, the detection results were consistent with sex of infants after delivery; the RASSF1A was amplified all in samples of other 9 cases after BstU1 cleavage, which confirmed existance of cffDNA. The ABO gene detection of cffDNA in plasma showed that out of 20 samples, both non O and B were amplified simultancously in 8 cases, suggesting the B blood group; the non O was amplified, but the B was not amplified only in 5 cases, suggesting A blood group, the non O and B both were not amplified in samples of 7 cases, suggesting O blood group. The above-mentioned detection results were consistent with new born ABO blood group by serological test.

CONCLUSIONThe proposed protocol for the detection of fetal ABO based on RASSF1A gene by using fetal DNA from maternal plasma can be used for noninvasive prenatal diagnosis of fetal ABO blood group.

ABO Blood-Group System ; Blood Grouping and Crossmatching ; DNA ; Female ; Fetus ; Genotype ; Humans ; Pregnancy ; Real-Time Polymerase Chain Reaction ; Tumor Suppressor Proteins

OBJECTIVETo investigate the expression of placenta-derived RASSF1A gene in maternal plasma during first and second trimesters, and to explore its value for the prediction of pre-eclampsia.

METHODSFor 325 pregnant women of the first trimester, free DNA of plasma samples was extracted at 7-12, 13-18, and 19-24 gestational weeks, respectively. Methylation-sensitive restriction enzyme digestion followed by fluorescence quantitative PCR (MSRE+ PCR) was employed for analyzing the concentrations of hypermethylated RASSF1A gene. Blood pressure, proteinuria and clinical feature were monitored at the same time. Those who had subsequently developed pre-eclampsia were selected as the pre-eclamptic group, 30 normal pregnant women were selected as the control group. Hypermethylated RASSF1A gene in maternal plasma was retrospectively analyzed. The relationship between clinical classification, type of pre-eclampsia and concentrations of the gene were further analyzed.

RESULTSTwenty-six out of the 325 pregnant women developed pre-eclampsia as their only complication. At 13-18 gestational weeks, the mean concentrations of fetus-specific RASSF1A sequences were 141.62 copies/mL in maternal plasma of pre-eclamptic pregnancies, which was significantly greater than that of the controls (98.90 copies/mL). Fetus-derived RASSF1A levels were 2.03 fold higher in pre-eclamptic subjects than controls at 19-24 gestational weeks. There was a significant difference in the level of hypermethylated RASSF1A gene between the mild and severe pre-clamptic subjects at 13-24 gestational weeks (P< 0.05). The concentrations of the sequences were significantly higher in early-onset severe pre-eclampsia than late-onset severe pre-eclampsia at 19-24 gestational weeks (P< 0.05).

CONCLUSIONAltered expression of hypermethylated RASSF1A gene may be detected in maternal plasma during second trimester, which has important significance for early prediction of pre-eclampsia.

Female ; Gestational Age ; Humans ; Placenta ; metabolism ; Pre-Eclampsia ; blood ; diagnosis ; genetics ; metabolism ; Predictive Value of Tests ; Pregnancy ; Pregnancy Trimester, Second ; Prenatal Diagnosis ; methods ; Tumor Suppressor Proteins ; blood ; genetics

OBJECTIVETo investigate the methylation in promtor region of RASSF2 and sFRP1 in sporadic colorectal cancer patients in order to provide screening method for early colorectal cancer.

METHODSThe methylation in promoter region of RASSF2 and sFRP1 in serum samples of 59 sporadic colorectal cancer patients and 59 healthy volunteers was detected by methylation specific PCR. The association between clinicopathological features of sporadic colorectal cancer patients and methylation in promoter region of RASSF2 and sFRP1 was analyzed.

RESULTSThe methylation rates of RASSF2 and sFRP1 gene in serum of 59 sporadic colorectal cancer patients were 27.1% and 30.5%, significantly higher than those in healthy volunteers(0%, both P<0.01). The methylation of RASSF2 or sFRP1 occurred in 29(49.2%) patients, which was significantly higer than the methylation rate of single gene(P<0.05). No association was found between methylation ratio of RASSF2 and sFRP1 and clinicopathological features in sporadic colorectal cancer patients.

CONCLUSIONSMethylation in promoter region of RASSF2 and sFRP1 is often detected in serum of colorectal cancer patients. The combination detection of methylation for the two genes may provide information for early screening of colorectal cancer.

Colorectal Neoplasms ; diagnosis ; genetics ; DNA Methylation ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; blood ; genetics ; Male ; Membrane Proteins ; blood ; genetics ; Middle Aged ; Promoter Regions, Genetic ; Tumor Suppressor Proteins ; blood ; genetics

OBJECTIVETo investigate the level of hypermethylated ras association domain family 1A (RASSF1A) gene in maternal plasma of pre-eclampsia and its clinical value.

METHODSSixty pre-eclampsia women including 30 mild and 30 severe cases were selected, 60 women with normal pregnancy were studied as control. Free DNA from plasma samples was extracted, fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the concentrations of RASSF1A gene before and after methylation-sensitive restriction digestion. Meanwhile, beta-actin gene was detected as a control to confirm complete enzyme digestion.

RESULTSThe median concentration of hypermethylated RASSF1A gene was 3.31-fold higher in samples from pre-eclamptic pregnancies than that in controls. There was significant difference between the mild and severe pre-eclamptic subjects (P<0.05), with the median concentrations of 1659 copies/mL and 2036.50 copies/mL, respectively.

CONCLUSIONHypermethylated RASSF1A gene in pre-eclampsia plasma was significantly increased and the concentrations were related to the severity of pre-eclampsia.

Adult ; DNA ; blood ; metabolism ; DNA Methylation ; Female ; Humans ; Pre-Eclampsia ; genetics ; metabolism ; pathology ; Pregnancy ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult

OBJECTIVETo assess the correlation of O6-methylguanine-DNA methyltransferase (MGMT) to radiation sensitivity of nasopharyngeal carcinoma (NPC).

METHODSEighty randomly selected NPC patients were divided into high (+/++, n=62) and low (-/+/+, n=18) MGMT groups according to the results of MGMT detection using immunohistochemistry. All the patients received irradiation with external beam radiotherapy, and the radiation sensitivity of NPC was analyzed after the irradiation.

RESULTSThe rates of high and low radiation sensitivity were 83.3% and 16.7% in low MGMT group, respectively, showing significant differences from those of the high MGMT group (45.2% and 54.8%, respectively, chi(2)=4.393, P=0.036).

CONCLUSIONThe content of MGMT correlates to the radiation sensitivity of NPC and may serve as valuable indicators for predicting the radiation sensitivity of NPC.

Adolescent ; Adult ; Aged ; Carcinoma, Squamous Cell ; enzymology ; radiotherapy ; DNA Modification Methylases ; blood ; DNA Repair Enzymes ; blood ; Female ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; enzymology ; radiotherapy ; Radiation Tolerance ; physiology ; Tumor Suppressor Proteins ; blood ; Young Adult