The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype–genotype correlation

Abstract

To compare single-molecule real-time technology (SMRT) and conventional genetic diagnostic technology of rare types of thalassemia mutations, and to analyze the molecular characteristics and phenotypes of rare thalassemia gene variants, we used 434 cases with positive hematology screening as the cohort, then used SMRT technology and conventional gene diagnosis technology [(Gap-PCR, multiple ligation probe amplification technology (MLPA), PCR-reverse dot blot (RDB)] for thalassemia gene screening. Among the 434 enrolled cases, conventional technology identified 318 patients with variants (73.27%) and 116 patients without variants (26.73%), SMRT identified 361 patients with variants (83.18%), and 73 patients without variants (16.82%). The positive detection rate of SMRT was 9.91% higher than conventional technology. Combination of the two methods identified 485 positive alleles among 49 types of variant. The genotypes of 354 cases were concordant between the two methods, while 80 cases were discordant. Among the 80 cases, 76 cases had variants only identified in SMRT method, 3 cases had variants only identified in conventional method, and 1 false positive result by the traditional PCR detection technology. Except the three variants in HS40 and HBG1-HBG2 loci, which was beyond the design of SMRT method in this study, all the other discordant variants identified by SMRT were validated by further Sanger sequencing or MLPA. The hematological phenotypic parameters of 80 discordant cases were also analyzed. SMRT technology increased the positive detection rate of thalassemia genes, and detected rare thalassemia cases with variable phenotypes, which had great significance for clinical thalassemia gene screening.

Case Report: The third-generation sequencing confirmed a novel 7.2 Kb deletion at β-globin gene in a patient with rare β-thalassemia

“…Furthermore, it was difficult to studies of molecular structure of these large deletions especially the accurate breakpoints, until the invention of TGS technology which could relatively accurately estimate the deletion range. TGS conducting on the PacBio sequencing platform, had shown significant advantages such as extra-long reads (80 Kb) and no requirement for PCR, had been used for thalassemia carrier screening in the last 2 years ( Xiao and Zhou, 2020 ; Liang et al, 2021 ; Luo et al, 2022 ).…”

Analysis of rare thalassemia genetic variants based on third-generation sequencing

“…Through third-generation sequencing for full-length of HBA1 , HBA2 and HBB genes, multiple variants may be recognized in one sample. Thus, it is crucial to evaluate the correlation between genotype and phenotype to further identify clinically significant variants 25 . The SNVs reported in this study, including HBA2 SNVs (c.168dup, c. − 59C > T, c.51G > T, c.91_93delGAG, and c.300 + 34G > A) and HBB c.316–45G > C, all showed some evidence related to abnormal hematology phenotypes and hemoglobin results.…”

Applications of next generation sequencing in the screening and diagnosis of thalassemia: A mini-review

“…Therefore, best practice recommends that NGS to always be paired with Gap-PCR to compensate for this shortcoming, and Sanger sequencing to remain a mandatory confirmatory test ( 64 ). Further, the advent of long molecule third-generation sequencing technology may also offer a similar solution by directly reading the entire length of the gene sequence with no apparent GC preference ( 65 , 66 ). Additionally, since NGS is moving rapidly into diagnostic laboratories, and novel variants are progressively added into the HbVar database, new variants of uncertain significance (VUS) will definitely be encountered ( 31 ).…”