ABSTRACT
AIM:To establish a kind of simple and efficient method for cell-free fetal DNA ( cff-DNA) enrich-ment and to investigate its range of applications and the advantages and disadvantages.METHODS:(1) The single nucleo-tide polymorphisms( SNPs) , which linked to paternalβ-thalassemia mutations, were screened.We analyzed the contact be-tween the SNPs inβ-thalassemia gene ( HBB gene) and haploid type by the Haploview software, and then selected these close SNPs which have higher heterozygosity with the HBB gene.(2) We selected 4 cases of different β-thalassemia muta-tions with their husband, and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma.If the IVS-II-654 mutation was not detected, we detected the SNP which linked to the IVS-II-654 mutation.Similarly, we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma.At the same time, we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma.RESULTS:(1) Nine cases of the SNP ( rs7480526) linked to the mutation at IVS-II-654 in HBB gene, and 11 cases of the SNP ( rs10768683) linked to the muta-tion at CD41-42 in HBB gene were detected.( 2 ) We detected 1 case who inherited the paternal β-thalassemia mutation (IVS-II-654).We did not directly detect patermal IVS-II-654 mutation in maternal plasma, but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100%detection, and 2 cases inherited the paternal β-thalassemia muta-tions (CD41-42) in the maternal plasma by TT-FULL-COLD-PCR and had 100%detection.However, we detected nothing by conventional PCR.CONCLUSION:TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.
ABSTRACT
Interleukin 6 (IL6) plays an essential role in the regulation of immune response to chronic disease. In this study, the three known single nucleotide polymorphisms (SNPs) in the IL6 promoter region were genotyped in a large chronic hepatitis B cohort to evaluate the effects of IL6 promoter variants. The single base extension method was used for this genotyping. Haplotypes were constructed by the three SNPs in IL6. Allele frequencies were compared for; i) patients with chronic hepatitis (CH) and chronic carriers vs. chronic hepatis patients with clinical evidence of liver cirrhosis (LC) (i.e., portal hypertension), ii) cirrhotic patients with hepatocellular carcinoma (HCC) vs. without HCC by logistic regression, and iii) with respect to the time intervals from the onset of infection to HCC. Results were analyzed by Cox relative hazard analysis on the assumption that all the patients were infected during early infancy. The frequencies of each SNP were 0.002 (IL6-597 G>A), 0.25 (IL6-572 C>G) and 0.002 (IL6-174 G>C), respectively, in the Korean population (n = 1,046). No significant associations were detected between IL6-572 C>G and chronic hepatitis B outcome in this study; i.e., chronic hepatitis B outcome in this study; i.e., LC occurrence on CH (OR = 0.16-1.27, P = 0.13- 0.71) and HCC occurrence on LC (OR = 1.04-1.23, P = 0.89-0.60) of heterozygotes and homozygotes for G allele in referent comparison to homozygotes for common allele (C/C genotype), and time interval to HCC (RH = 0.67-1.00; P = 0.14-0.99). In conclusion, there appeared to be no significant associations between IL6 promoter variants and disease outcome in chronic hepatitis B.