Identification Of A De Novo HBB Frameshift Mutation Causing β-thalassemia Major

BackgroundThalassemia is one of the most common single-gene genetic diseases,which can be divided into two categories:α-thalassemia and β-thalassemia.Generally speaking,β-thalassemia is an autosomal recessive inherited disease,which only shows hematological abnormalities in carriers,and is the most severe in homozygous and compound heterozygous patients and relies on regular blood transfusions.Dominantly inherited β-thalassemia are a group of conditions,in which the patients inherit a single β-thalassemia allele and normal α-globin genes at the molecular level,result in intermedia or major anemia.This has aroused our thinking and attention to dominant β-thalassemia.In this study,we studied a new mutation in the HBB gene which causedβ-thalassemia major phenotype and analyzed its molecular characteristics.Materials and methods1.Subject:Our proband is a three-year-old boy from Taizhou,Zhejiang,China.He presented with unexplained severe anemia and splenomegaly.2.Methods:Blood samples of proband and their family members were collected for hematological analysis,isopropanol test and inclusion body test.The new mutation allele was captured by next-generation sequencing,and then primers were designed to amplify the segment and the PCR products were verified by Sanger sequencing.The paternity test was used to prove the biological relationship between the proband and his parents.Hair follicle and oral swab samples of the proband and ther father’s semen were collected to extracte DNA for chimerism analysis.Species conserved analysis of the mutant sites and prediction of the secondary structure and three-dimensional structure of the protein were performed.293T cells were transfected with HBB wild type and HBB:c.399delA mutant vectors.Meanwhile,qPCR was performed to analyze the expression of HBB in cells.ResultsNext-generation sequencing and Sanger sequencing confirmed that the proband carried a new unreported frameshift mutation HBB:c.399delA.Hemoglobin electrophoresis did not detect abnormal hemoglobin in the proband,and isopropanol test and Heniz body were negative.The parents and the older brother did not carry the new mutation.Paternity test confirmed that the proband had inherited two sets of genetic material from each parent.The HBB genotypes of the proband’s hair follicles and mouth swabs were consistent with the blood samples,and the new mutation was not detected in the protester’s father’s semen.Species conservation analysis suggested that the sequence was highly conserved.The protein structure prediction results indicated that the HBB:c.399delA β-globin was extended by 10 amino acids at the carboxyl terminus.In vitro experiments confirmed that mutant HBB:c.399delA could affect the translation of β-globin gene.ConclusionsHere,we present a case of dominant β-thalassemia caused by frameshift mutations in HBB exon 3.Our study demonstrates the importance of reporting new mutations and potential mechanisms for dominant β-thalassaemia,extending the mutant spectrum of β-thalassaemia and getting further understanding the heterogeneity of the disease.