Analysis Of Genetical Cause Of Three Hereditary Hemolytic Anemia Families

Hereditary hemolytic anemia（HHA）is a group of hereditary diseases caused by the mutation of genes encoding erythrocyte membrane protein,erythrocyte enzyme or hemoglobin.HHA is characterized by shortened life span of red blood cells.Its clinical manifestations are mainly hemolytic anemia and hemolytic anemia with varying severity.In hereditary hemolytic anemia,thalassemia is a common hemoglobinopathy,and deficiency of glucosephosphate isomerase（GPI）is a rare deficiency of erythrocytase in the glycolysis metabolic pathway.This research is divided into two parts,the identification of disease genes and genetic research were carried out in a family with rare GPI deficiency and two families with thalassemia respectively.This extended the genetic spectrum of the two HHA,providing new experiences and ideas for the diagnosis of HHA.Part Ⅰ Identification of pathogenic gene in a family with glucosephosphate isomerase deficiencyBackground Glucosephosphate isomerase（GPI）deficiency is a rare erythrozyme deficiency disorder caused by mutations in the GPI gene encoding glucosephosphate isomerase and inherited in an autosomal recessive pattern.The main clinical manifestations are chronic non-spherocytic hemolytic anemia,and a few patients are accompanied by neuromuscular symptoms.Due to its non-specific phenotype and rare reports,clinical diagnosis is difficult.In this study,a patient with GPI deficiency misdiagnosed as thalassemia was corrected and further genetic analysis and diagnosis were performed.Methods The peripheral blood of family members was collected,and genetic analysis was performed by hematological phenotype analysis,thalassemia related mutation analysis,whole exon sequencing,pathogenic site screening and Sanger sequencing verification.Then the diagnosis was confirmed by bioinformatics prediction of mutation sites,family members’ medical history and clinical phenotypic analysis and related experiments.In addition,the literature related to GPI mutation was summarized.Results The proband carried two novel mutations in the GPI gene,c.1207C>T and c.1255G>C,inherited from father and mother,respectively,and carries a thalassemia mutation,HBB:c.126₁29delCTTT,inherited from father.The activity of GPI enzyme in red blood cells of both the proband and the mother was reduced to varying degrees.Combined with clinical phenotype and genotype,the proband was identified as GPI deficiency.Subsequently,the reported GPI deficiency patients with genotypic validation were summarized through literature search,and the GPI gene mutation profile was plotted.Conclusion This study describes the clinical features and diagnostic process of a patient with rare GPI deficiency,correcting her misdiagnosis of thalassemia.Two novel mutations of GPI gene were found,which enriched the genetic spectrum of GPI gene.The rare HHA occurring in the thalassemia endemic region is more likely to be misdiagnosed as thalassemia due to the similarity of clinical phenotypes.Accurate diagnosis depends on more comprehensive etiological cognition and consideration.Part Ⅱ Identification of two families with copy number variation ofα-globin gene leading to thalassemiaBackground Thalassemia is a common inherited blood disorder caused by mutations in or the loss of genes that regulate globin synthesis.One of the genetic causes is copy number variations（CNVs）in the α-globin gene cluster.However,there is no unified summary and discussion on the detailed information and mechanisms of these CNVs.In this study,two novel CNVs were identified in two Chinese families with diagnosed cases of thalassemia.Methods We investigated these two families by hematologic analysis,routine genetic testing for thalassemia,multiplex ligation dependent probe amplification（MLPA）,next-generation sequencing（NGS）and other methods.In addition,all known CNVs involving the α-globin gene were summarized and the generation mechanism of these genome arrangements were analyzed systematically.Results Two novel CNVs involving the α-globin gene,a duplication（αααα159）and a deletion（--259）,were identified in two patients with β-thalassemia intermedia and Hb Bart’s hydrops fetalis syndrome,respectively.Through literature reviews,42 CNVs involving the α-globin gene were selected for analysis.Based on the different molecular characteristics of the breakpoint region,we suggest that the occurrence of these CNVs can be attributed to several possible mechanisms.Conclusions This study described two novel CNVs involving the α-globin gene,which extend the variation spectrum of globin genes.In addition,explorations of the generation mechanism of these CNVs provides a mechanistic perspective for the molecular etiology of thalassemia.