Study On Molecular Diagnosis Technology Of Thalassemia Mutation Based On SNPscan And CNVplex Technology Molecular Genetics Of Hb H Disease Caused By Hb Agrinio Combined With Southeast Asian Type Deletion

Background and objectivesThalassemia?thalassemia?is characterized as hypochromic microcytic hemolytic anemia as a result of globin gene defects inducing hematopoietic failure.As it's an autosomal recessive genetic disease,high-risk couples both carrying ?-or ?-globin mutation have a chance of giving birth to a Hb Bart's or Hb H hydrops fetalis,or offsprings with thalassemia major or intermedia.The current therapy for thalassemia is regular transfusion combined with iron removal by iron chelating agents,which casts a heavy economic burden and great mental pressure on the family and the society for its big expense.Prevention of thalassemia outweighs treatment.Hence,it's an important part of the public health to carry out the population census,the genetic counseling,and comprehensively popularize premarital and prenatal examinations to prevent the birth of Hb Bart's hydrops fetalis and children with intermediate or severe thalassemia.According to the latest HbVar data?http://globin.bx.psu.edu/cgi-bin/hbvar?,more than 500 types of thalassaemia-related mutations have been found worldwide,including?--SEA/?,the commonest alpha deletion in the Chinese population,?-?3.7/?,?--?4.2/?,?--THAI/?,?--FIL/?,?--11.1kb/?and?-?27.6kb/?,etc.The most common point mutations are??WS?/?,??Qs?/?,??CS?/?.In addition,there are??CD31?/?,and??CD78?/?,etc.There are at least 54 types of ? thalassemia point mutations reported in Chinese population,of which the following eight mutations account for more than 93%:HBB:c.126₁29delCTTT,HBB:c.52A>T,HBB:c.316-197C>T,HBB:c.-78A>G,HBB:c.216₂17insA,HBB:c:79G>A,HBB:c.92+1G>T and HBB:c.-79A>G.In addition,there are HBB:c.-123A>T,HBB:c.-140C>T and HBB:c.162delT,etc.There have been 5 types of deletional ?-thalassemia reported in Chinese population,including Chinese deletion,Southeast Asian HPFH deletion,Taiwanese deletion,Gantonese deletion and Yunnanese deletion.The current gene detection methods for point mutations causing thalassemia are PCR-RDB,Sanger sequencing,fluorescent PCR melting curve assay,HPLC,HRM,and ARMS-PCR.The detection methods for deletional thalassemia include Southern blot,Gap-PCR,MLPA,real-time PCR,and array-CGH.They can be used in routine clinical testing,but they are not suitable for gene screening in large populations owing to their cumbersome operation and low detection throughput.Besides,the popular second-generation sequencing can be used for gene screening of large populations with the advantages of high accuracy,sensitivity and throughput in recent years,but it is difficult to be applied on a large scale due to its high cost.The establishment of a simple,rapid,accurate,reliable,economical and practical high-throughput detection technology can not only meet the public health needs of large-scale population screening,but also present a new challenge to the genetic diagnosis technology for thlassemia.The goal of this study is to establish a new method for molecular diagnosis of thalassaemia based on the same detection platform.It is accurate,reliable,simple and practical,high-throughput,and low-cost and suitable for large-scale population screening and routine molecular diagnosis of thalassemia.Participants and methodsAccording to the pathogenesis and molecular basis of thalassaemia,the following research protocols have been specifically implemented targeting the ?/?thalassaemia point mutations and deletion mutations that have been reported?Up to the start of this study?in the Chinese population.1.SNPscan technology to detect thalassemia point mutations-The highly specific ligase was used to recognize the allele of the SNP sites,and non-specific sequences with different lengths were added to at the ends of the ligation probes and then the ligation products with various lengths corresponding to different loci were obtained through ligation reaction.Subsequently,the ligation products were amplified by the universal primers labeled with fluorescences followed by capillary electrophoresis to separate sequences with different fluorescences.Finallly,the electrophoresis map was analyzed to genotype different SNP loci.2.CNVplex technology to detect thalassemia deletional mutations-The ligation probes with non-specific sequences with different lengths were hybridized with the target fragments and ligated by the ligation reaction based on highly specific ligase,thus producing ligation products with different lengths corresponding to different sites.And then the ligation products were amplified by universal primers labeled with different fluorescences.and the PCR products were separated by capillary electrophoresis according to different lengths of the product.At last,the capillary electrophoresis spectrum was analyzed and the copy number of the target gene were calculated by the peak value ratio of target gene and the reference gene.3.Preliminary evaluation of the diagnostic method.To provide a simple and practical molecular diagnostic method for the current large-scale screening of thalassaemia is one of the goals of this study.In order to initially evaluate the practicability and feasibility of the new diagnostic method for screening of thalassemia,100 samples for pre-marital or prenatal detection were selected and tested by the novel methd,gap-PCR,and Sanger sequencing simultaneously to compare the test results.4.Evaluation of the diagnostic method by blind analysis of large samples.1747 samples with complete phenotypic data were prepared,whose genotypes have been confirmed by gap-PCR,RDB,and Sanger sequencing.These samples were blindly numbered and tested and analysed by the novel assay.Then the samples with unmatched results compared with known genotypes were re-detected comparatively by gap-PCR or Sanger sequencing to comprehensively evaluate the sensitivity,accuracy and practicability of the new method for molecular diagnosis of thalassaemia.ResultsAccording to the research objectives,the research results obtained by the implementation of specific research programs were as follows:1.A stable and reliable SNPscan detection technology for thalassemia point mutations was established.This system was based on the genetic analysis system of ABI 3130xl PRISM.It was divided into two panels.PanelAl could detect 38 different thalassaemia mutation sites,while panelA2 could detect 29 different thalassaemia mutation sites.And its specifity was verified to be reliable.2.A stable and reliable CNVplex detection technology for thalassemia deletion mutations was established.The system was based on the genetic analysis system of ABI 3130xl PRISM,and the gene copy numbers of HBB,HBAl and HBA2 were detected to diagnose the deletional thalassaemia.And its specifity was verified to be reliable.3.In the preliminary evaluation experiment,the new method,gap-PCR and Sanger sequencing were used to simultaneously detect 100 gDNA samples for antemarital or prenatal examination.And 16 cases of ?-thalassemia heterozygotes and 5 cases of ?-thalassemia heterozygotes were detected.The detection results of the two methods were consistent,and mutations checked out were common genotypes in Chinese population.In conclusion,the accuracy of the new method were 100%.The new technology was simple,easy to implement,high-throughput,and accurate and reliable,preliminarily demonstrating the feasibility and practicality of the new method for large-scale population screening.4.Evaluation of the diagnostic method by blind analysis of large samples:1747 samples were composed of 100 normal samples,636 H disease samples?31 samples compounded with ?-thalassemia heterozygous mutations?,1011 samples with intermediate/severe ?-thalassemia?155 samples compounded with mild/still type?-thalassemia and 5 compounded with Hb H disease?.Comparing the detection results of SNPscan and CNVplex assay and traditional methods,the results of 1737 samples were consistent,and 10 samples had different results.After further verification,the results of the 10 samples were consistent with the results of SNPscan and CNVplex.It could be concluded that the accuracy of SNPscan and CNVplex detection methods was 100%in detecting thalassemia point mutations and deletions,and the method was stable and reliable.ConclusionThis study established a SNPscan and CNVplex method to detect thalassemia point mutations,deletions or duplications.The SNPscan detection method can detect 67 point mutation sites of ?-/?-thalassemia,and the CNVplex detection method can detect deletional ?-/?-thalassemia.Through a comprehensive evaluation of sensitivity and accuracy,and a preliminary evaluation of the practicability of population screening,the new molecular diagnostic method established in this study is fully confirmed to be accurate,reliable,simple and practical,high-throughput,low-cost,and suitable for large-scale screening screening and routine diagnosis of thalassaemia.SNPscan assay has the advantages of high throughput,simple operation,high accuracy,low cost,and short time-consuming.CNVplex assay has the advantages of high throughput,simple operation,high accuracy,and low cost.It is suitable for clinical genotyping and even the genetic screening of large populations.Background and objectivesAlpha thalassemia is a genetic hemolytic anemia caused by mutations of the alpha globin gene that completely or partially inhibits the synthesis of the alpha globin chain.It is one of the most common single-gene genetic diseases in southern China.The disease is mainly caused by the deletion of alpha globin gene,and a small part is caused by point mutation of alpha globin gene.Combined with hematological phenotype and globin genotype data,an accurate diagnosis was performed in a case with severe anemia in Guangdong.Participants and methodsThe peripheral blood of the family members of the proband was collected,and the red blood cell parameters and hemoglobin components were analyzed by a fully automatic three-class blood analyzer and HPLC hemoglobin analysis system.The genomic DNA was extracted by the traditional phenol-chloroform method,and the family was genotyped by Gap-PCR,RDB and Sanger sequencing.ResultsHematological phenotype showed that the proband had severe anemia of small cell hypopigmentation?MCV60.6pg,MCH18.1fL,Hb50g/L?and abnormal hemoglobin H positive was detected by hemoglobin component analysis;the proband's grandmother phenotype was normal;the proband's parents and uncle showed small cellular hypopigmentation.Genetic analysis revealed that the genotype of the proband was--SEA/??CD29,the father's genotype was--SEA/??,and the mother and uncle genotypes were aaCD29/??.ConclusionHb Agrinio?HBA1:CD29 T>C?is an unstable abnormal hemoglobin,and the hemoglobin component is negative,which must be diagnosed by genetic analysis.The mutation complex ?0-thalassemia manifests as Hb H disease,and the degree of anemia is usually heavier.