Study On Novel Mutation Of Hereditary Spherocytosis And The Diagnostic Value Of Eosin-5-maleimide Binding Test

Background and ObjectiveHereditary spherocytosis?HS?is a type of chronic hemolytic disease associated with congenital defects in the erythrocyte membrane caused by gene mutation of regulated proteins and approximately 75% patients are autosomal dominant for the disease.HS is found globally at different prevalence in different countries.The prevalence of HS is as high as 1/2000 in northern Europe and North America.Although anemia,jaundice,and splenomegaly are typical clinical manifestations of HS,due to the different types of membrane protein defects,clinically the disease is still highly heterogeneous.In about 20-30% of HS patients,clinical manifestations are not obvious,except for a compensatory increase in reticulocytes?RET?.In these patients,hemolytic symptoms are aggravated when certain factors are induced,the most common of which are infections or continuous heavy physical activities.Moreover,severe HS patients often present with severe hemolysis,and require blood transfusions to maintain a hemoglobin?HGB?content of >60g/L.However,this procedure can often lead to iron overload or other complications in these patients.Although HS is not an uncommon clinical disease,its definitive diagnosis rate remains relatively low.Highly diverse type of clinical manifestations towards the disease is one of the main reasons of misdiagnosis and mistherapy of many HS patients.Identification of a suitable laboratory test has important implication in the improvement of HS diagnosis.In recent year,eosin-5'-maleimide?EMA?binding test has become a novel screening method for the diagnose of HS,which advantages of high sensitivity,small sample amount needed,fast detection,simple procedure and so on have attracted much attention.EMA binding test has been widely applied in abroad while few investigations have been reported about internal research.In this study,EMA binding test was used for re-assessment of already diagnosed with HS to validate the diagnostic value of the technology.Then,systematic analysis of HS patient and his family was carried out to explore the relationship between gene mutation form of HS and clinical phenotypic heterogeneity,developing the relevant gene mutation spectrum.Methods36 HS patient from 20 different families who were diagnose in the First Affiliated Hospital Guangxi Medical University was collected.DNA from peripheral blood was extracted the proband and her family members according to reagent box explanation and amplified by PCR.Gene mutations were determined using sequence capture array & solexa sequencing and the detected mutations were further verified by sanger sequencing.Point to the novel mutation that have never been found in the gene database,prediction of harmful effects was taken.To exclude genetic polymorphisms,newly-identified mutations were subjected to large-scale gene screening using high resolution melt analysis.Protein expression levels in the erythrocyte membrane of the proband were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?and Western blot?WB?analysis.The type of protein defect in the extracted RBC membrane proteins was confirmed by western blot?WB?.In this study,we selected three family to report.At the same time,52 patients who were diagnosed as Mediterranean anemia and 198 tests who were physical examination qualified in the First Affiliated Hospital Guangxi Medical University were collected.Exam them with EMA binding test,evaluate the diagnostic value of the method by statistical analysis the sensitivity and specificity.ResultsThe sensitivity is 94.4% and the specificity is 97.2% when use percentage reduction of mean channel florescence%?MCF%?>14.32% as the optimum cut-off point when evaluate the diagnostic value of EMA binding test.In the three selected HS family,both parents of proband with no clinical symptoms of anemia,jaundice,and splenomegaly brings about negative family history.Mutation were all found in spectrin protein.Proband 1 was ?-spectrin partial deficiency and c.161A>C,c.5572C>G,6531-12C>T were respectively detected in exon 2,exon 40 and intron 45 of SPTA1 gene.Proband 2 was ?-spectrin partial deficiency and c.2226₂239del mutation was detected in exon 13 of SPTB gene.Proband 3 was ?-spectrin partial deficiency and c.2179₂180del,c.3710A>G mutation were respectively detected in exon 16,exon 26 of SPTA1 gene.Conclution1.It is successful that applying EMA binding test to diagnose HS patient.When use percentage reduction of mean channel florescence%?MCF%?>14.32% as the optimum cut-off point,the sensitivity is 94.4% and the specificity is 97.2%.2.Combined with laboratory test data and pedigree findings,three proband were diagnosed with HS.HS has high diversity of clinical manifestation,which is similar with other reports.3.We firstly reported that 4 novel mutations from three different families.?1?c.161A>C heterozygous mutation,which results that histidine was replaced by proline.?2?c.2226₂239del deletion mutation,which results premature termination of amino acid coding,producing truncated proteins.?3?c.2179₂180del deletion mutation,which results premature termination of amino acid coding,producing truncated proteins.?4?c.3710A>G heterozygous mutation,which results that arginine was replaced by glutamic acid.