Application Study Of High-resolution Melting Analysis In Genetic And Prenatal Diagnosis Of Thalassemia

α-thalassemia is a kind of hemolytic anemia inherited in autosomal recessive mode. Itresults from deletions or point mutations of alpha globin genes leading to reduced orabsent synthesis of alpha globin chain. The three most common deletional mutations in theChinese population are--SEA,-α^3.7,and-α^4.2, and the three most common non-deletionalmutations are Hb Constant, Spring （Hb CS）, Hb Quong Sze,（Hb QS） and Hb Westmead（Hb WS）. Hemoglobin Bart ’s edema fetal can not survive after the birth, and the severestform of α-thalassemia compatible with postnatal life is hemoglobin H（Hb H） disease. HbH disease concludes two types: deletional Hb H disease and nondeletional Hb H disease.Patients with nondeletional Hb H disease have more severe clinical manifestation thanthose with deletional Hb H disease. Therefore, we should pay particular attention to avoidthe missing in diagnosis of non-deletional Hb H disease. Currently the Hb CS hemoglobincould be screened by capillary electrophoresis, but not Hb QS and Hb WS. α-thalassemiacarriers with only one point mutation, are usually asymptomatic, and the routine bood testand hemoglobin analysis are normal. Therefore, it may cause the missing in screening ofα-thalassemia （except of Hb CS）. In prenatal screening, it is necessary to screen the pointmutations of alpha globin gene in individuals whose partners are a carrier ofα⁰-thalassemia. Several methods can be used to detect non-deletion α thalassemiamutations, including reverse dot blot, allele-specific PCR and sequencing, however eachhas its own limitations. In this study, we applied high-resolution melting analysistechniques to detect the three common point mutations of alpha-thalassemia in Chinesepopulation, and evaluate its value in clinical application. ObjectionTo detect the three common point mutations of alpha-thalassemia in the Chinesepopulation by high resolution melting analysis. To establish a simple, low-cost, reliableand high-throughput screening method.Methods1. Sample collection: during December2008to October2011, a total of81DNA sampleswere collected with point mutations of alpha-thalassemia, genotyped by gap-PCR, reversedot blotting and DNA sequcencing previously.34males,44females with age rangingfrom4days to50years old were included. In addition,3prenatal diagnosis samples wereincluded. During July to September2011, a total of14DNA samples were colleted asnormal controls including5males,9females with age ranging from22to48years old.Normal controls were confirmed by sequencing without α （α1and α2） globin gene pointmutations. All the samples were collected from prenatal diagnostic center of Guangzhouwomen and children’s medical center.2. PCR amplification: Primers were designed by LightScanner Primer Design softwarewhich could amplify the third exon of α₁and α₂globin genes simultaneously, covering HbConstant Spring, Hb Quong Sze and Hb Westmead. PCR system were established andoptimized.3. HRM analysis: after PCR amplification, the PCR products were analyzed by HRMusing LightScanner96. Melting curves were analyzed by LightScanner Software withCall-IT2.0.ResultsFive types of melting curves were identified by HRM analysis:（1）Hb QS(α^QSα/αα)；（2）Hb QS with–SEAor-α^4.2(α^QSα/--SEA、α^QSα/-α^4.2)；（3）Hb CS or Hb CSwith deletioal mutations(α^CSα/αα、α^CSα/--SEA、α^CSα/-α^4.2、α^CSα/-α^3.7)；（4）Hb WS or HbWS with–SEAor-α^3.7(α^WSα/αα，α^WSα/--SEA，α^WSα/--α^3.7)；（5）normal controls（αα/αα）.All the samples with point mutations of alpha-thalassemia could be correctly distinguished from the normal controls, and Hb QS can be distinguished from Hb QS with deletionalmutations. In the present study, the sensitivity and specificity of HRM analysis were100%for detection of Hb Constant Spring, Hb Quong Sze and Hb Westmead.ConclusionHRM is a simple, accurate, rapid, high-throughput and cost-effective geneticsanalysis technique, it could be used as an optimized method for identification of the threecommon point mutations of alpha-thalassemia in Chinese population. β thalassemia is one of the most common monogenic disease in the word. βthalassemia was caused by mutations in β globin gene and inherited in an autosomalrecessive mode. So far, more than200kinds of β thalassemia mutations have beenidentified, of which five mutaitons （CDs41-42-TCTT, IVS2-654C> T,-28A> G, CD17A> T, CDs71-72） accounted for90%of all mutations. The children withsevere β thalassemia require regular blood transfusions after birth, and most fetus will diein young, unless the hematopoietic stem cell implement is performed successfully.Therefore, prenatal diagnosis is the preferred method to prevent the birth of children withsevere β thalassemia. The methods to detect β thalassemia mutations includeallele-specific PCR, reverse dot blot, denaturing high performance liquid chromatographyand so on. In this study we applied high resolution melting analysis to detect βthalassemia mutations in the Chinese population, and to explore its clinical value.ObjectionTo detect the common β thalassemia mutations in Chinese population by HRM. Toestablish a simple, efficient, low cost, accurate, reliable and high-throughput molecularscreening method, and to explore its application in prenatal diagnosis of β thalassemia.Method1. Sample collection: DNA samples from30couples with high-risk β thalassemia and their fetal were collected from prenatal diagnostic center in Guangzhou women andchildren’s medical center. The samples were genotyped by reverse dot blot and sequencingpreviously. A total11kinds of common β globin gene mutations were studied:-29A>G，-28A>G， CDs14-15+G， CD17A>T， CD26G>A， CDs27-28+C， IVS1-1G>T，CDs41-42-TCTT， CD43G>T， CDs71-72+A， IVS2-654C>T. Of the30prenatalsamples, there are13villus samples,16amniotic fluid cell samples and1cord bloodsamples. In addition,3normal control samples were collected, which were confirmedwithout HBB gene mutations by sequencing.2. PCR amplification:4pairs of primers were designed by LightScanner Primer Designsofeware to amplify the first exon, the second exon and part of the second intron （coveringIVS2-654-C> T mutation） of HBB gene. PCR systems were established and optimized.3. HRM analysis: after PCR amplification, the PCR products were analyzed by HRMusing LightScanner96. Melting curves were analyzed by LightScanner Software withCall-IT2.0.ResultThe genotypes of the fetus were compared with the melting curve of theirparents（positive control） and normal controls. Of the30fetus,10fetus were diagnosed asserve β thalassemia patients,11cases were diagnosed as a β thalassemia carriers, and9cases as normal. In this study, the results of HRM were consistent with that ofreverse dot blot and sequencing.ConclusionHRM analysis is an effective, low-cost and accurate method, and could be used forrapid prenatal diagnosis of β-thalassemia with known genotypes in parents.