PMC10020366_01
Thalassemia genetic testing by LRS technology. (A) Pedigree of the family. (B, C) Integrative Genomics Viewer plot displayed the CCS reads of SMRT sequencing for the proband (II-1) and mother (I-2). The pink area showed the alphaalpha allele, and the green area showed the other allele with 14.9-kb deletion. (D) Validation of the large deletion by gap-PCR. The primers were designed flanking hg38 chr16:168803-183737 to detect the 14.9-kb deletion. (E,F) Confirmation of the exact breakpoints of deletions by Sanger sequencing for the proband (II-1) and mother (I-2). (G) Sequence of breakpoint junctions of the 14.9-kb deletion. Reference sequences encompassing the breakpoints were shown above and below the sequences with deletion. The sequences with microhomology in the breakpoint junctions were underlined. (H) Display of the 14.9-kb deletion in related to other recurrent deletions in the alpha-globin gene cluster.
fgene-14-1156071-g001.jpg
PMC10020366_01_fgene-14-1156071-g001.jpg
F1
CBC and Hb analysis were also performed for his parents (Figure 1A). Hybyibio Limited Cororation, China), and PCR-RDB technology (Chaozhou Hybyibio Limited Cororation, China) were performed following the kit's instructions for 23 most common thalassemia variants, including three deletional variants of alpha-thalassemia (--SEA, -alpha3.7, and -alpha4.2), three non-deletional variants of alpha-thalassemia (Hb Constant Spring (Hb CS, HBA2:c.427 T>C), Hb Westmead (Hb WS, HBA2:c.369C>G), and Hb Quong Sze (Hb QS, HBA2:c.377 T>C)), and 17 common beta-thalassemia variants (-28 (HBB:c.-78A>G), -29 (HBB:c.-79A>G), -30 (HBB:c.-80 T>C), -32 (HBB:c.-82C>A), CD26 (HBB:c.79G>A), CD31 (HBB:c.94delC), CDs14-15 (HBB:c.45_46insG), CD17 (HBB:c.52A>T), CDs27-28 (HBB:c.84_85insC), CDs41-42 (HBB:c.124_127delTTCT), CD43 (HBB:c.130G>T), CDs71-72 (HBB:c.216_217insA), IVS-I-1 (HBB:c.92 + 1G > T), IVS-1-5 (HBB:c.92 + 5G > C), IVS-II-654 (HBB:c.316-197C > T), Int (HBB:c.2 T > G), and CAP (HBB:c.-50A>C). However, the PCR-RDB assay did not identify any variants (Figure 1B), which suggested that the microcytic hypochromic anemia in the proband and his mother might be caused by a rare thalassemia variant beyond the detecting scope of the PCR-RDB assay.