Identification Of Rare Variants In α-and β-thalassemia Individuals Using Third-generation Sequencing And Optical Mapping Technology

Background:Thalassemia is one of the most common genetic diseases in the world,which is caused by point mutation and deletion of globin gene.Accurate genotyping of thalassemia is the basis for genetic counseling and prenatal diagnosis.At present,routine detection of α-and β-thalassemia methods and reagents only include more than 20 common mutation types in China.Traditional screening methods have limitations and can only detect gene mutations with limited scope.Encountering rare mutations in thalassemia may lead to erroneous results and misdiagnosis in clinical diagnosis.TGS based on long-read single-molecule sequencing detects complex thalassemia gene structural variants with long read-length and high accuracy,and can detect point mutations and complex structural variants in globin genes at the same time.628 patients were tested for thalassemia genes,including those with different results or phenotypes that did not match the genotype in routine thalassemia tests.We applied single molecule real-time(SMRT)sequencing using third-generation sequencing after multiple long-range PCR and identified 15 rare mutations,as well as a tandem repeat mutation containing 24 additional HBA2 genes using Bionano optical mapping(BOM)technology.Methods:In this study,628 patients with thalassemia underwent combined hemoglobin testing and routine hematology examinations,and screening and molecular diagnosis of thalassemia on collected samples.Finally,we sent all 628 patient samples to an independent laboratory for SMRT sequencing.One case of tandem repeat mutation with high copy number was detected using optical mapping.Subsequently,the phenotype of the obtained rare mutation samples was analyzed and elaborated.Results:Among the 628 enrolled cases,SMRT and BOM identified 576 patients(91.72%)with α-thalassemia,17 patients(2.71%)with β-thalassemia,35 patients(5.57%)with compound α and β-thalassemia were identified.Among them,SMRT identified 31 variant types.Among them,SMRT detected 31 types of mutations,and optical mapping technology identified 1 type of tandem repeat mutation.A total of 1289 positive alleles were detected.A total of 15 rare and 1 novel clinically significant variants were discovered.The qPCR and reversed-phase high-performance liquid chromatography detection were performed on family members with HBA2 duplication mutations.It was found that the genes in the duplication mutations did not express,and also led to a decrease in mRNA expression and the proportion of αglobin peptide chain.Conclusion:Our results showed that SMRT has great advantages in detection of α-globin gene triplications,rare deletions and determination of a cis or trans configuration.SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis,especially for detection of rare thalassemia mutations.This case report highlighted the important role of structural organization of the globin locus in gene activity,which would provide a new clue to investigate the distal enhancer and globin gene promoter communication during erythroid development.