Study On The Application Value Of The Third Generation Sequencing Technology In Thalassemia Gene Detection And Prenatal Diagnosis

Objective:To evaluate the clinical application value and feasibility of the third generation sequencing technology in thalassemia gene detection and prenatal diagnosis by comparing the results of the third generation sequencing technology with conventional and high-throughput sequencing technology.To analyze the hematological phenotypes of rare variants detected in this study,and to provide reference for clinical genetic counseling.To summarize the variation types of thalassemia detected by the third generation sequencing technology in Jiangxi region,and enrich the thalassemia gene mutation spectrum in this region.Methods:1.A total of 402 patients who were screened positive for thalassemia in Jiangxi Provincial Maternal and Child Health Hospital from October 2019 to October 2022 were selected for thalassemia gene detection using the third generation sequencing technology.Among them,319 cases were synchronously tested by conventional technology(Gap-PCR,PCR-RDB)and 83 cases were synchronously tested by highthroughput sequencing technology for thalassemia gene detection.The gene detection results and positive detection rate of the third generation sequencing technology and these two technologies were compared,and the hematological phenotype of rare variants detected during the study was analyzed.The variation types of thalassemia in the region detected by the third generation sequencing technology were analyzed statistically.2.From January 2020 to December 2020,40 high-risk families who received prenatal diagnosis of thalassemia by conventional techniques(Gap-PCR,PCR-RDB)in Jiangxi Provincial Maternal and Child Health Hospital because both spouses carried identical thalassemia genes were successively included.The third generation sequencing technology was used to retrospectively analyze the amniotic fluid genotypes of pregnant women.The genetic test results were compared with those detected by conventional technique.Results:1.Among the 402 screened positive subjects,4.08%/2.41% more subjects were detected to carry thalassemia gene by the third generation sequencing technology than the conventional technology(319 cases)and the next generation sequencing technology(83 cases),and 18 /4 more gene variants were detected by the third generation sequencing technology.In addition,Gap PCR and Sanger sequencing were used to verify the 18 cases /4 cases with inconsistent gene detection results of the two technologies.The verification results were consistent with the result of third generation sequencing technology,and the hematological phenotypes of rare mutation carriers detected by the third generation sequencing technology were analyzed.In addition,the third-generation sequencing technology detected a total of 47 variants,including 31 thalassaemia variants,in 402 subjects.In addition to the 23 α-and β-thalassaemia variants that are common in clinical practice,the third generation sequencing technology also detected 5 rare α-and β-thalassaemia variants with clear pathogenicity,3 neutral polymorphic loci and 5 variants of unknown pathogenicity.2.In 40 high-risk families with prenatal diagnosis of thalassemia,30 fetal thalassemia genes were accurately detected by both the third generation sequencing technology and conventional technology,and the positive detection rates and genetic test results of the two technologies were completely consistent.Conclusions:1.In the gene detection of thalassemia,the third generation sequencing technology has a higher positive detection rate than conventional technology and high-throughput sequencing technology,and can additionally detect rare variants with diverse phenotypes and complex structural variants.The third generation sequencing technology can accurately distinguish the highly homologous genes of HBA1/2,which is of great value for the clinical gene detection of thalassemia.However,the pathogenicity of many rare mutations is still unclear and needs further family analysis,which brings great challenges to clinical genetic counseling.2.In the prenatal diagnosis of thalassemia,the detection accuracy of the third generation sequencing technology is 100%.The third generation sequencing technology can detect the full length of α and β globin gene at the same time,and the detection range is broader and more comprehensive than the conventional technology.The third generation sequencing technology does not need to extract amniotic fluid DNA and PCR amplification,which reduces the possible contamination and simplifies the process,and is expected to be applied to the prenatal diagnosis of thalassemia.