ABSTRACT
Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.
ABSTRACT
Objective To explore the relationship between the HLA-G 14 bp insertion/deletion polymorphism and the infection of Epstein-Barr virus(EBV) for children.Methods The study genotyped HLA-G 14 bp insertion/deletion polymorphism of 102 infectious mononucleosis children and 165 normal controls by PCR-PAGE,detected the plasma sHLA-G level of 51 infectious mononucleosis children and 146 normal controls by ELISA.Results A significant difference was observed for the frequencies of the HLA-G 14 bp genotype between the two groups( x2 =6.742,P=0.034 ),and a significant difference was also observed for the 14 bp allele frequencies between the two groups( x2 =6.672,P=0.01 ).The plasma sHLA-G levels in the infectious mononucleosis children were dramatically higher than that in normal controls,and a significant difference was observed between the two groups( Z=-9.472,P＜0.01 ).Among the infectious mononucleosis children,levels of sHLA-G was find a significant difference between the three genotypes of HLA-G 14 bp insertion/deletion polymorphism( H=6.09,P =0.048 ),and the level of s HLA-G with 14 bp+/+ genotype was markedly lower than that of the two other genotypes (Z=-2.376,P=0.01 8).Conclusion There was a relationship between the HLA-G 14 bp insertion/deletion polymorphism and the susceptibility to the infectious mononucleosis for children.Children who carried the 14 bp-/- genotype or deleted the 14 bp allele may have a significantly increased risk of the infection of EBV.The plasma sHLA-G might be considered as an index for auxiliary diagnosis infectious mononucleosis.