Evaluating the Clinical Utility of a Long-Read Sequencing-Based Approach in Prenatal Diagnosis of Thalassemia

Abstract

Background The aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia. Methods A total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses. Results Among the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from β-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from β-thalassemia trait to intermedia and changed the disease prognosis. Conclusions CATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.

Case report: A novel 10.8-kb deletion identified in the β-globin gene through the long-read sequencing technology in a Chinese family with abnormal hemoglobin testing results

“…Rare deletions, triplets, quadruples, and other unreported variants could be missed. A long-read sequencing-based approach termed CATSA has been established for identifying both α- and β- thalassemia genetic carrier status, whose clinical utility has also been extended successfully to a diagnosis of thalassemia ( 9 , 10 , 18 ). A novel heterozygous 10.8-kb deletion on chromosome 11 was identified through CATSA and was confirmed by MLPA and Gap-PCR.…”

Third generation sequencing transforms the way of the screening and diagnosis of thalassemia: a mini-review

“…Despite its significant technical advantages, the high equipment costs and long turnaround times of PacBio limit its integration into routine screening and diagnostic procedures. While the total cost of preparing TGS libraries and sequencing reagents for each sample has dropped to below $20 ( 21 ), mainstream PacBio sequencing equipment is still expensive (e.g., the PacBio Sequel II device costs approximately $350,000), limiting its adoption by many medical institutions. If TGS library preparation samples are sent to biotech companies for sequencing, the overall testing cycle takes about 8 days (including sample transportation time) in China, whereas the testing cycle for traditional methods is no more than 3 days ( 21 ).…”

“…While the total cost of preparing TGS libraries and sequencing reagents for each sample has dropped to below $20 ( 21 ), mainstream PacBio sequencing equipment is still expensive (e.g., the PacBio Sequel II device costs approximately $350,000), limiting its adoption by many medical institutions. If TGS library preparation samples are sent to biotech companies for sequencing, the overall testing cycle takes about 8 days (including sample transportation time) in China, whereas the testing cycle for traditional methods is no more than 3 days ( 21 ). It is expected that PacBio will release sequencing equipment that covers medium and low throughput with greatly reduced costs, and this equipment can fully meet the low throughput testing requirements for thalassemia currently.…”

A Feasibility Study of Non-Invasive Prenatal Testing of Thalassemia by Haplotype Analysis Based on Next Generation Sequencing and Long-Read Sequencing

“…This was done as described in (Liang et al 2023) with modi cations.The gDNA samples were rst subjected to multiplex long PCR using optimized primers to produce speci c amplicons that encapsulate currently known structural variation regions, SNVs, insertions and deletions in the HBA1, HBA2, and HBB genes. After puri cation and end repair, double barcode adapters were ligated to the 5' and 3' ends.…”

“…Recently, long-read thirdgeneration sequencing (TGS) has emerged as a powerful tool to detect the genetic basis of α-and β-thalassemia, especially for detecting complex structural variants and CNVs. TGS offers a convenient way to directly analyze parental haplotypes by utilizing ultra-long read lengths of up to 30 kb-100 kb, providing high accuracy (QV30 > 99.9%), single-molecule resolution, and no GC preference (Liang et al 2023;Liu 2022). This technology may improve the detection rate of NIPT in thalassemia.…”