Clinical Genotyping By NGS Reveals A De Novo Dominant HBB Exon Mutation Causing Abnormal Hemoglobin

Hemoglobinopathies are the most common monogenetic disease globally and account for approximately 3.4%of deaths in children under 5 years of age.Hence this inherited blood hemoglobin(Hb)disorders are great challenges for human health.This group of diseases are further classified into two categories:abnormal Hb and thalassemia.Abnormal Hb is one of the most common hemoglobinopathies worldwide.There are already 1,771 entries of Hb variants and thalassemias in the HbVar database,and over 1,300 globin gene mutations are associated with abnormal Hbs including the famous HbS which causing sickle cell disease.Different Hbs could lead to symptoms at different levels.Many Hb variants usually have no clinical symptoms,while some Hb variants might causing elevated oxygen affinity and subsequently leading to erythrocytosis.And some Hb Variants are unstable,abnormal Hbs causing hemolytic anemia.Less commonly,some abnormal Hbs could lead to a decreased oxygen affinity.Thalassemias are prevalence inherited hemolytic disorders worldwide.Clinically,thalassemia is classified into a-thalassemia and β-thalassemia,which are caused by mutations in the a-globin gene cluster and the β-globin gene cluster,respectively.Among them,β-thalassemia is caused by mutations in the β-globin gene cluster,which leads to a group of hematological disorders.The form of the hemoglobin tetramer varies with different human developmental stages.There are three types of hemoglobin in the embryonic stage:Hb Gower-1(ζ2ε2),Hb Gower-2(α2ε2),and Hb Portland(ζ2β2).The fetal hemoglobin composition is HbF(a2y2),while in adulthood,in addition to main composition HbA,there is a small fraction of HbA2(α2δ2).Several clinical studies have shown that elevated levels of fetal hemoglobin can alleviate the severity of β-hemoglobinopathies,including β-thalassemia and sickle cell anemia.In this paper,we report a de novo mutation in the exon 3 of HBB produces abnormal Hb identified in a girl originated from Chongqing,China.Our proband was evaluated for hemolytic anemia following her first transfusion at Beijing Children’s Hospital at her age of four.This clinical diagnosis showed splenomegaly,jaundice and hemolytic anemia without the evidence of iron,vitamin B12,or folate deficiency.To investigate further,blood samples from proband’s parents and proband were sent to our lab.Proper informed consent obtained from the parents was also sent to us.Similar to diagnostic results from Beijign Children’s Hospital,Red blood cell count parameters showed that proband had macrocytic,hemolytic anemia with an elevated HbF,and HPLC showed no abnormal Hb was identified.Traditional genotype for common thalassemia showed all three samples were negative for those common mutations in the a-and β-globin genes identified in the south Chinese popultaion.Proband samples was further analyzed by our previously designed NGS panel and high-through sequencing data revealed that a de novo nucleotide deletion was present in our proband at nucleotide position 408 in exon 3 of the HBB gene(HBB:c.408delT),but absent in either of her parents.After Sanger sequencing,paternity and maternity test,we conclude this is a de novo mutation.We did a secondary structure prediction for our proband’s abnormal hemoglobin chain and found that C-terminus of this hemoglobin chain was elongated and disrupted by aβ-sheet.What’s more,we found an HbF-associated rSNP which is reported before in the HBG1 of the proband that might be the major factor responsible for proband’s elevated HbF levels.Through this study,we expand the the spectra of Hb variants and provide strong evidence for accurate diagnosis and precision medicine.