Assessment of global DNA methylation in stem cells with reverse-phase high performance liquid chromatography.

Objective: We tested the possibility of the use of reverse-phase high performance liquid chromatography (RP-HPLC), a sensitive, simple and cost effective technique, for evaluation of global DNA methylation alteration in stem cells. Methods: We detected genomic methylation in four cell lines of mouse embryonic stem cells and in the cell at various developmental stages, including mouse embryonic fibroblast (n=5) and mouse tissues (n=9), using RP-HPLC. The samples were extracted for single nucleosides and investigated for the concentrations of deoxy-cytidine (dC), deoxy-guanosine (dG) and deoxy-methyl-cytidine (5-dmC) using RP-HPLC. The methylation levels were obtained by a ratio of the entire genomic 5m-C to dC. Results: Our results demonstrated that the RP-HPLC technique can be used for analysis of global methylation on the stem cell genome. It can display the discrepancy of methylation levels among the cells at different degrees of cell differentiation. Conclusion: We present the evidence that the RP-HPLC technique can be used for the entire-genomic methylation detection in stem cells and should be further developed as a strategy for monitoring the global DNA methylation of stem cells in advanced research.

Analysis of polymorphisms in the 5'-flanking region of the apolipoprotein(a) [Apo(a)] gene in Thai subjects with coronary artery diseases.

Lipoprotein(a) [Lp(a)] is a complex lipoprotein particle in human plasma. It is composed of apolipoprotein B (Apo B)-100 and apolipoprotein(a) which are linked by a disulfide bond. Plasma levels of the Lp(a) vary greatly (over 1,000 folds) among individuals. Elevated plasma levels of the Lp(a) have been shown to be an independent risk factor for coronary artery diseases (CAD). The level of Lp(a) is controlled by a single gene, the Apo(a) gene, with multiple alleles; each encodes different concentrations of the Lp(a). Previous studies revealed the presence of polymorphisms in the 5'-flanking region (FL) of the Apo(a) gene at 3 positions: G or A (-914), C or T(-49), and G or A (-21), which can be detected by cleavage of PCR-amplified DNA products with TaqI, MaeII and HhaI, respectively. The 5'-FL genotypes of the Apo(a) gene can be classified by the combination of the presence (+) or absence (-) of these restriction sites into 5 types; type A, +++, type B, -++, type C, -+-, type D, --+ and type E, +-+. In the present study, the authors analyzed the 5' FL types of the Apo(a) gene by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 100 healthy control subjects, 26 CAD patients with [Lp(a)] < or = 30 mg/dL, and 94 CAD patients with [Lp(a)] > 30 mg/dL. The authors found that the genotype frequencies of the Apo(a) gene were 53, 16, 27 and 4%, for types A, B, C and D respectively in normal healthy controls. In CAD patients with [Lp(a)] < or = 30 mg/dL, the distribution of the genotype frequencies were 53.8, 11.5, 30.8 and 3.9% for types A, B, C and D, respectively. Additionally in CAD patients with [Lp(a)] > 30 mg/dL, the genotype frequencies were 60.6, 11.7, 21.3 and 6.4% for types A, B, C and D, respectively. The present study might shed some light to understand CAD at the molecular level.

Glutathione and glutathione peroxidase in type 2 diabetic patients.

Objectives: Hyperglycemia is claimed to cause oxidative stress in diabetic (DM) patients. The influence of free radical production by hyperglycemia may exacerbate the cardiovascular complications in diabetes. However, the effects of glycemic control and cardiovascular complications is diabetes on oxidative stress parameters have been not fully studied. This study compared the red cell glutathione (GSH) level and glutathione peroxidase (GPx) activity in fairly controlled typed 2DM(Fasting plasma glucose {EPG}-<180 mg/dl) poorly-controlled type 2 DM (FPG>180 mg/dl), and type 2 DM complicated with coronary heart disease (CHD) with that in a normal healthy group (FPG <110 mg/dl). Materials & Methods : GSH level and GPx activity were determined in the red cells of 19 subjects with poorly controlled type 2 DM, 26 subjects with fairly controlled type 2 DM, and 20 subjects with type 2 DM complicated CHD with that of 20 healthy subjects with normal plasma glucose level matched for age and gender who served as a control group. In all groups of DM these oxidative stress parameters were compared to a control group by one-way ANOVA test. The association between these parameters and FPG was to a control group by one-way ANOVA test. The association between these parameters and FPG was determined using the Pearson product moment correlation. Results : The red cell GSH levels were significantly lower in all types of diabetes compared with those of age-matched normal control subjects (p<0.05).Red cell GPx activity was significantly increased only in the poorly controlled type 2 DM and type 2DM with CHD (<0.05). The decrement of red cell GSH may be due to the higher consumption of GSH for the increasing of GPx activity or there is a reduction or the pentose phosphate pathway that stimulated by insulin resulting in lowered GSH recycling. The association between FPG and GSH or GPx activity in all subgroups of type 2 diabetic patients compared with normal healthy subjects showed no correlation. Conclusion : These findings suggested that type 2 DM patients were susceptible to oxidative stress and higher blood glucose level in poorly controlled type 2DM and type 2 DM complicated with CHD had an association with free radical-mediated lipid peroxidation. Therefore, any means that can reduce oxidative stress may be beneficial for the prevention or slowing of progression of cardiovascular complication in these types of diabetic patients.

Studies of GH gene defects in familial Isolated Growth Hormone Deficiency (IGHD).

One family with isolated GH deficiency (IGHD) was Studied. To determine GHI gene deletions, PCR and Southern blot analyses were used. None of the possible deletions were found in the subjects but the GH1 gene mutation was found. The family consisted of parents (both 140 cm) and their three children with isolated GHD. The daughter and two sons were first seen between 2.9 and 5.3 years of age when their HtSDSs were -2.2 to -3.6, and peak GHs were 0.9 to 4.0 mg/ml. The GH1 gene change was found in G-->A substitution at +28 in the intervening sequence or intron 3 (IVS3+28 G-->A). This change is a dominant-negative mutation which has never occurred in any reports in any reports in Thailand and we were the first group to report here. The segregation which and expression studies of the IVS3+28 G-->A variant are underway to confirm whether it is a new dominant-negative mutation that causes GHD by perturbing mRNA splicing.

Molecular studies in growth hormone gene deletions in patients with isolated growth hormone deficiency.

Human GH deficiency (GHD) occurs in 1 in 4,000-10,000, and up to 30 percent of cases have an affected first degree relative suggesting many cases may be familial. To determine the GHD cases cased by GH gene defects we analyzed the GH genes of 90 consecutive cases. Genomic DNAs were used for PCR amplification of 2.7 kb fragments containing subjects’ GH gene; these PCR products were subjects to determine GH gene deletions. PCR products of 1900 and 1919 bp were obtained. By using the combination of restriction enzymes BglI, HaeII and SmaI to digest these PCR products, the various sizes of GH gene deletion can be detected. None of the possible deletions was found in these patients by either PCR or Southern blot analysis.

Determination of the apolipoprotein E genotype using PCR-RFLP in kidney diseases.

Using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), genotypes of the apolipoprotein E (Apo E) were determined in 50 control subjects and 60 Thai kidney diseases patients including nephritic syndrome and chronic renal failure (CRF) (30 each). Exon 4 of the Apo E genomic DNA was amplified between nucleotide numbers 2849 and 3071 (270 base pairs) then digested with HhaI. It is observed that E3/3 was the most common genotype found in the control subjects (80%). In nephritic syndrome patients, E4/3 was found to be the most frequent (53.3%). On the contrary, E3/3 was found to be the most prominent in CRF patients (80%). There was a significant different of the Apo E genotype in hephrotic syndrome from the normal control subjects (p < 0.05 by X2 analysis). One the other hand, there was no significant difference of the Apo E genotype in CRF patients from the control subjects (p>0.5). Cholesterol and triglyceride levels of the E4/3 nephrotic syndrome patients were significantly different from the normal controls of the same genotype (p<0.05). Similarly, in CRF patients, triglyceride level of the E3/3 genotype was also significantly different from the normal controls of the same genotype (p<0.05). These results suggested that polymorphism of the Apo E genotypes may be associated with the lipid abnormalities in renal diseases.