Noninvasive Prenatal Testing Of Thalassemia Based On Target Capture Next-generation Sequencing

Objective:Thalassemia is one of the most common monogenic inherited disease with carrier frequency of 10%in South of China.Blood transfusion therapy or bone marrow transplantation can only be used.However,it is expensive and cannot be completely cured.Therefore,prenatal screening and diagnosis are of great significance for the prevention of thalassaemia.Currently the first-choice method to prevent the birth of the affected fetal by prenatal genetic diagnosis in high-risk couples.Given the fact that the invasive prenatal diagnosis can only be performed by professionals in experienced centers,and it may increase the risk of miscarriage or infection.The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive prenatal.diagnosis.Since the cell-free fetal DNA was discovered in maternal plasma,the non-invasive prenatal diagnosis has been rapidly developed.With the continuous optimization of Next Generation Sequencing and the development of bioinformatics,The application of NIPT from T21,T18,T13 chromosomal aneuploidy detection,extend to copy number variation disease research,showing its wide application prospects and clinical value.The cell-free fetal DNA based NIPT is safer and easily acceptable.It is likely to establish a safe,and non-invasive method for prenatal of thalassemia in the future.Methods:Eight families diagnosed as carriers either alpha-thalassemia or beta-thalassemia.All families are from The Third affiliated hospital of Sen-Yat sun university.The maternal plasma and amniotic fluid were collected for prenatal diagnosis.The genome DNA of the parents and amniotic fluid was multiple-PCR amplified by the thalassemia panel,and the cell-free DNA was captured by a set of thalassemia probes.The thalassemia panel and probes were designed in the targeting exons of the HBA,HBB and the highly heterozygous SNPs within the 2Mb flanking region.The Ion AmpliSeqTM thalassemia Panel was composited by Life Technologies,consiting of four primer pools made up of 1354 pairs a-thalassemi primers and 361 pairs 0-thalassemia primers.And the products were sequenced by the next-generation sequencing.Further,then the parental haplotype was constructed by the trios-based strategy.The fetal paternal haplotype was detected by SNP from cell free fetal DNA,then the fetal maternal haplotype was deduced from the Relative Haplotype Dosage(RHDO)and Sequential Probability Ratio Test(SPRT).Results:1.Only 5 mL peripheral blood or 2 mL plasma is needed for capture sequencing,noninvasive prenatal testing of thalassemia can be achieved.2.Comparing the calculation of fetal concentration based on the SNP ratio and fetal Y chromosome concentration,the difference was not statistically significant(P>0.05).3.The parental genotypes and haplotypes were successfully deduced in 8 families.4.The fetals' genotypes and haplotypes were successfully deduced in 8 families analysed with RHDO and SPRT.5.The noninvasive prenatal testing of thalassemia were identical to the invasive prenatal diagnosis results with an accuracy rate of 100%.Conclusion:Our study demonstrates that the effective noninvasive prenatal testing of thalassemia with the next-generation sequencing.This study shows that NGS is a rapid,high-throughput,high-sensitivity detection method,showing great advantages and feasibility in the noninvasive prenatal testing of monogenic diseases.