Neonatal Screening For Alpha-thalassemia In Guangzhou District And The Application Of High-resolution Melting Analysis In Screening For Nondeletion Type Of Alpha-thalassemia

Alpha-thalassemia usually results from deletions or mutations of α-globin gene. Itis one of the most commonly inherited single-gene disorders in the world. Guangzhou,a city of South China, has a high population frequency of α-thalassaemia. The normalown four alpha genes. If all α-gene deletion(Hb Bart’s hydrops foetus syndrome), thefetus usually dead in late pregnancy. In addition, patients with non-deletional Hb Hdisease or non-deletion α-thalassemia homozygous have more severe anemia, even needblood transfusion. Parents with silent α-thalassemia and α-thalassemia trait have noclinical manifestation themselves, but they may conceive hydrops foetus or babies withtransfusion-dependent α-thalassemia. So newborn screening should be taken to preventbirth defects and improve the quality of newborn babies. Some studies have shown that,Hb Bart’s of umbilical cord blood is related to α-thalassemia. This syudy use automaticcapillary electrophoresis (CE) to determinate Hb Bart’s quantitative of umbilical cordblood for screening α-thalassemia, explore the effective of this method. Besides,high-resolution melting (HRM) is an emerging genetic approach in resent years. Thismethod has the advantages of rapid, economical and closed-tube, this study Explore thefeasibility of using HRM to detect the point mutation of non-deletion α-thalassemia.Objective1. To investigate the prevalence of α-thalassemia and frequencies of genotypes innewborns in Guangzhou District.2. To investigate the correlation between the levels of Hb Bart’s of cord blood and α-globin genotypes.3. Explore the feasibility of using high-resolution melting to detect the point mutationof non-deletion α-thalassemia.Methods1. During May2010to July2011,6525cases of umbilical cord blood were collectedafter delivery at Guangzhou Women and Children Hospital.2. Automatic capillary electrophoresis system (the Capillarys2)(Sebia, Paris, France)was used to determinate Hb Bart’s amount in cord blood of6,525newborns.3. Samples with the presence of Hb Bart’s should be done genetic diagnosis throughgap-PCR to detect the most common deletional α-thalassemia:--SEA、-α3.7and-α4.2.100cases of negative samples(without Hb Bart’s) were selected randomly for the genetictesting.4. For the samples with Hb Bart’s%≧2%but the results of gap-PCR are nomal,use aMultiplex PCR to detect four kinds of deletional α-thalassemia:--THAI,--FIL,--MED,-(α)20.5.5. Singled out the samples with Hb Bart’s≤2.5%but the results of gap-PCR andMultiplex PCR are nomal,use high-resolution melting to screening for the third exon ofα-globin gene.6. To the samples above, use reverse dot blotting technique to detect the most commonpoint mutation of non-deletion α-thalassemia: αCSα、αQSα and αWSα.7. For samples of unknown genotype, use high-resolution melting to screening for thefirst two exon of α-globin gene.8. Do gene sequencing of the whole α1-globin gene and α2-globin gene for the samplesof unknown genotype.Results1. Totally,377samples were found positive for Hb Bart’s; among these,375sampleswere confirmed by DNA testing, including14genotypes with384α–thalassemiaalleles. There are12samples with αCSα, accounted for0.18%,10samples with αQSα, accounted for0.15%, and the--SEAaccounted for4.03%.2. The amount of Hb Bart’s was increased with the increasing numbers of the defectiveα-globin genes. Newborns with one α-globin gene defect have Hb Bart’s at0.55%±0.29%, while Hb Bart’s levels in individuals with two α-globin genes defectare3.54%±0.79%, and HbH disease patients have Hb Bart’s at20.27%±1.72%.3. After Gap-PCR,1case of-α3.7/αα was found between the100cases of negativesamples (without Hb Bart’s).4. Both sensitivity and specificity of useing HRM analysis to Screening fornon-deletional α-thalassemia are100%.5. After gene sequencing of the whole α1-globin gene and α2-globin gene, four kindsof non-deletion α-thalassemia were detected, and two of which are novel.Conclusion1. This study confirmed a high population frequency of α–thalassaemia in GuangdongDistrict.2. Hb Bart’s in umbilical cord blood as a indices, has a high efficiency and specificityin screening for Hb H desease、α0–thalassaemia and αCSα/αα, but Hb Bart’s is notsuitable as the screening indices for diagnosis of α+-thalassemia except αCSα/αα.3. To determinate Hb quantitative, CE has the advantages of convenient, effective,economical and accurate, can be used to screening for α-thalassemia.4. HRM is a rapid, cheap and closed-tube method with high sensitivity and specificityin scanning for non-deletion α-thalassemia.