The Effect Of Second Generation Sequencing Technology In Children With Genetic Refractory Hemolytic Anemia

Objective: To analyze and research the etiology and pathogenesis of 38 children with hemolytic anemia,to provide theoretical foundation for the causes,diagnosis,treatment and prognosis of refractory hemolytic anemia.Through further analysis of specific cases to establish effective ways provides the reference and optimization approach for the diagnosis of refractory hemolytic anemia.Methods: Thirty eight patients with hemolytic anemia hospitalized in Changhai hospital from January of 2016 to December of 2016 were preliminary diagnosed through detailed medical history collection and physical examination,routine examinations,Coomb’s test,bone marrow puncture and other relevant laboratory examinations.To those who can not clear the cause through routine examinations,we used a full test of hemolytic anemia on the red cell membrane,enzyme,and hemoglobin.When the etiology is still unknown or with relapsed anemia,the second generation sequencing technology was used in 15 cases to sequence 271 genes that related with hereditary anemia,and analysis the data with InDel analysis and SNP analysis.Then use generation sequencing technology to verify all the SNPs and InDels associated with disease which we filtered out.Meanwhile,in order to confirm the source of these mutations and genetics mechanism,we also sequenced the genetic mutations in part of the families with patients and analyze the data.Results: Through the analysis of 38 children with HA of etiology analysis,routine inspection,a full test on the red cell membrane,enzyme,and hemoglobin,we found that in 38 cases,18 cases were caused by infection(18/38,47.4%),including 2 EB virus infection and 2 mycoplasma pneumoniae infection,11 cases were caused by autoimmune hemolytic anemia(11/38,28.9%),cases were caused by red cell membrane disease,cases were caused by enzymatic abnormalities,cases were caused by hemoglobinopathy.The second generation sequencing technology were used in 15 cases to sequence 271 genes that related with anemia when the etiology is still unknown or clinical seizures recurrenced.Ten patients were identified with single genetic or Multi-gene mutations,including 12 missense mutations,1 nonsense mutation and 3 frameshift mutations.Positive rate was 66%.Thirteen mutations were firstly reported.The variation of conserved amino acid residues was caused by all mutations.Provean 、 SIFT and PolyPhen2 or Mutationtaster predicted these mutations were disease-causing.Conclusion: Genetic testing results show that single and compound gene mutations are common causes in children with refractory hemolytic anemia.The second generation sequencing technology is an effective and quick method to study etiology and genetic mechanism in hereditary hemolytic anemia.The results may also help us in clinical diagnosis and offering genetic counseling.