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.

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Objectives: To investigate the effect of HS-40 regulatory site deletion on α-globin gene expression and its clinical significance. Methods: Venous blood samples of subjects were analyzed using a hematology analyzer and high-performance liquid chromatography; fetal cord blood was analyzed by a capillary electrophoresis analyzer. Gap-polymerase chain reaction (PCR), reverse dot blot (RDB), and multiple-link-dependent probe amplification (MLPA) were used for genotyping of thalassemia. Results: The proband was POLR3 K, HS-40 heterozygous deletion; the proband's wife was -SEA/αα; the fetus was POLR3 K, HS-40 heterozygous deletion combined with -SEA deletion; all of them had microcytic hypochromic anemia. Fetal umbilical cord blood electrophoresis revealed a suspected Hb Bart's band to be 88.4%, and the fetus was, thus, diagnosed as Hb Bart's fetus. The red blood cell parameters of the sporadic case showed that he had microcytic hypochromic anemia. Hemoglobin (Hb) electrophoresis analysis showed Hb H to be 5.3%, leading to a diagnosis of Hb H disease. Gap-PCR and RDB identified the genotype to be -α3.7/αα, β A /β A . MLPA detected heterozygous deletion or -α3.7 deletion on one allele and deletion of the HS-40 regulatory site on the other allele. Conclusion:The deletion of HS-40 regulatory site reduced expression of α-globin. HS-40 heterozygous deletion manifested as mild anemia, which was of microcytic hypochromic type. When compounded with -α3.7/αα, it manifested as Hb H disease; and when compounded with -SEA/αɑ, it manifested as Hb Bart's fetus.

Conventional methods for the diagnosis of thalassemia include gap polymerase chain reaction (Gap-PCR), reverse membrane hybridization (RDB), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. In this study, we used single molecule real-time technology (SMRT) sequencing and discovered four rare variants that have not been identified by conventional diagnostic methods for thalassemia. We also performed genotype and phenotype analyses on family members of thalassemia patients. The SMRT technology detected five cases in which the proband had abnormal results by conventional diagnostic methods or inconsistencies between the genotype and phenotype. The variants included two cases of an α-globin gene cluster 27,311 bp deletion, --27.3/αα (hg38 chr16:158664-185974), one case of an HS-40 region 16,079 bp deletion (hg38 chr16:100600-116678), one case of a rearrangement of -α3.7α1α2 on one allele and one case of a ß-globin gene cluster HBG1-HBG2 4,924 bp deletion (hg38 chr11:5249345-5254268). This study clarified the hematological phenotypes of four rare variants and indicated the application value of SMRT in the diagnosis of rare α-globin and ß-globin gene cluster deletions, gene recombination and deletion breakpoints. The SMRT method is a comprehensive one-step technology for the genetic diagnosis of thalassemia and is particularly suitable for the diagnosis of thalassemia with rare deletions or genetic recombination.

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