A Study On The Gene Test And Prenatal Diagnosis For Thalassemias

Objective To set up stable, rapid and accurate assays which could be routinely used for detecting common a globin gene andβglobin gene mutations by applying multiplex PCR (mPCR) and PCR-reverse dot blot (PCR-RDB). To explore the importance of the gene test and prenatal diagnosis in thalassemias and the gene mutation types of both thalassemias in Yunnan populations.Methods 1. Multiplex PCR (mPCR) method based on Gap-PCR was performed for detecting three commonest deletion of a globin gene (--^SEA,-α^3.7 and -α^4.2) in southern Chinese populations. 2. PCR-RDB method was applied for testing three common types of non-deletionalα-thalassemia (α^CSα,α^QSαandα^WSα). 3. Eighteen types of most frequently mutations ofβglobin gene in south China were detected simultaneously with PCR-RDB method: CD41-42(-TCCT),IVS-2 nt654 C→T,-28 A→G,CD71-72 (+A),CD17A→T,CD26G→A,CD31(-C),CD27-28 (+C),CD43G→T,-32C→A,-29A→G,-30T→C,CD14-15 (+G),CAP,Int→IVS-1 nt1 (G→A, G→T)and IVS-1 nt5 G→C. 4. Prenatal diagnosis was carried out by the mentioned techniques above for fetuses who were in the risk of thalassemia major. 5. The relationship between genotypes and clinical features was analyzed, combining DNA sequencing when necessary to attain the precise diagnosis.Results 1. Among 107 cases who were suspected thalassemias, 33 cases of a-thalassemia and 40 cases of P-thalassemia were founded. Five genotypes were detected in 33 cases of a-thalassemia patients, including 3 cases of silent a-thalassemia(their genotypes are 2 cases ofαα/-α^3.7 and 1 case ofαα/-α^4.2), 23 cases of mild a-thalassemia (their genotypes are 22 cases ofαα/--^SEA and 1 case ofα^CSα/αα), 6 HbH patients (their genotypes are 2 cases of -α^3.7/--(SEA), 2 cases of -α^4.2/--^SEA, 1 case ofα^CSα/--^SEA and 1 case ofα^{Int ATG→A-G}α/--^SEA respectively), and 1 case of Hb Bart's hydrops fetalis syndrome (the genotype is --^SEA/--^SEA); Six genotypes were detected in 40 cases ofβ-thalassemia patients, including 3 cases ofβ-thalassemia major (their genotypes are 1 case of CD17 A→T/CD26 G→A, 1 case of CD71-72 (+A)/-28 A→G and 1 case of CD17 A→T/-28 A respectively), 1 case of intermediate P-thalassemia (the genotype is -28 A→G/CD26 G→A) , 36 cases of p-thalassemia traits (their genotypes are 15 cases of CD17 A→T/β^A,7 cases of CD41-42(-TCCT)/β^A,5 cases of -28 A→G/β^A,3 cases of CD26 G→A/β^A,3 cases of CD71-72 (+A)/β^A and 3 cases ofⅣS-2-654 C→T/β^A respectively) . 2. Prenatal diagnosis were supplied to 13 fetuses who were in the risk of thalassemia major, and revealed 1 case of intermediateα-thalassemia (HbH-CS patient), 2 case of mildα-thalassemia (their genotypes both areαα/--^SEA) and 3 cases ofβ-thalassemia heterozygote (their genotypes are CD17/β^A,CD41-42/β^A and -28/β^A respectively) , the others are not founded deletion or mutations. 3. Among thirty-three a-thalassemia patients, deletion form is the main form of gene mutation types and --^SEA is the commonest allele; in forty P-thalassemia patients, the most frequent gene mutation type is CD17, the second are CD41-42, CD26 and TATAnt-28.Conclusion 1. mPCR technique and PCR-RDB method can make a rapid and accurate gene test in detection ofα- orβ-thalassemia and these techniques may have good values to clinical application, but it seems that some cases could be missed. So it is necessary for making comprehensive studies with the relationships between genotypes, phenotypes and hematologic analysis, and DNA sequencing is complementally used in order to acquire the precise diagnosis. 2. Applying the methods mentioned to diagnose the fetuses who are in the risk of thalassemia major in prenatal diagnosis can prevent the births of these babies. 3. Comparing with other areas of China, the distribution features of gene mutations are speciality in Yunnan.