PMC9511401_01
Routine genetic assays for 23 types of variations in HBA and HBB genes. (A) Auspension-array system was performed to detect 23 types of common variations in thalassemia. (B) Reverse dot blot (RDB) assay was used to detect 23 types of common variations in thalassemia (PC was beta17M/N and alpha-SEA/N). No routine variants in thalassemia had been detected in this couple by this two independent platforms (NTC: No Template Control; PC: Positive Control; Pass: Quality Control Passed).
fgene-13-984996-g001.jpg
PMC9511401_01_fgene-13-984996-g001.jpg
F1
However, no variants had been detected of the above 23 genotypes of thalassemia in this couple (Figure 1).
MLPA and TGS results. (A) MLPA result showed the deletion across 10 probes targeting HBB gene. (deletion probes involved 10 probes between hg38 loc. 11p15.4: 5224905-5229846, including HBB-up, HBB-1, HBB-1 (WT) c., HBB-Intr.1, HBB-Intr.2, HBB-3 and HBB-down regions). (B) TGS result showed the exact length of the deletion was 7.2 Kb (Chr11:5222800-5230034, hg38) located at HBB gene.
fgene-13-984996-g002.jpg
PMC9511401_01_fgene-13-984996-g002.jpg
F2
HBB-up, HBB-1, HBB-1 (WT) c., HBB-Intr.1, HBB-Intr.2, HBB-3, and HBB-down regions, Figure 2A). However, the exact breakpoint location was not determined. TGS was adopted to characterize the deletion. Then, consistent with the MLPA result, TGS analysis confirmed a novel 7.2 Kb deletion (Chr11:5222800-5230034, hg38) at beta-globin gene, and this deletion removed all of the HBB gene (Chr11:5225464-5227197, hg38) (Figure 2B).
Gap PCR and Sanger sequencing results. (A) Gap PCR and agarose gel electrophoresis were performed to detect the deletion breakpoint. With the primers (7.2 Kb deletion forward primer: 5'-ATTTCTCCCTTGATAGTTTCTACTTTGGGTT-3', Wild Type forward primer: 5'-TACCATCATCCTGGCTTCAAGGC-3' and the common reverse primer: 5'-ATCTGAAGACTGTACCTCTGCTCTCC-3'), an unique fragment about 742 bp in length was only amplified in the husband (Normal: Normal individual; Blank: Water). (B) The fragment was then sequenced, the breakpoint was accordant with the TGS result.
fgene-13-984996-g003.jpg
PMC9511401_01_fgene-13-984996-g003.jpg
F3
Ultimately, this novel deletion was further verificated by Gap PCR and Sanger sequencing (Figure 3).