Gene Mutation Analysis In Chinese Patient With Hereditary Spherocytosis

Background and ObjectiveHereditary spherocytosis (HS) is a hereditary haemolytic disease. Theinheritance is dominant in about75%of cases, whereas the remaining are trulyrecessive cases and de novo mutations. The clinical manifestations of HS varywidely from asymptomatic carrier to severe haemolysis. The molecular basis ofHS involves mutations of the genes such as ANK1, SLC4A1, SPTA1, SPTB,EPB42, which lead to deficiency or absence of erythrocyte membrane proteins.The proteins that involved in the vertical interactions between the cytoskeletonand lipid bilayer result in reduced membrane surface area, a reduction in theratio of surface area to volume, and the formation of spherocytes with reduceddeformability that are selectively retained and damaged in the spleen.Researchers abroad have found many different mutations of EPB42. Althoughmany cases have been reported in china, but there has no report the EPB42genemutation by the method DNA direct sequencing, and relative mRNAquantification of the EPB42gene by the method of real-time PCR.We investigated relative mRNA quantification of the EPB42gene usingreal-time PCR, to select defects of erythrocyte membrane proteins. And to detect the mutations of EPB42using DNA direct sequencing. And then verifies thegenetic mutations in his parents. which identify pathogenesis and would furtherprovide scientific evidence for therapy and genetic counseling of the HS patientMethodsWe used sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and real-time PCR to identify the erythrocyte membrane proteinsdefect causing HS. Total genomic DNA was extracted from the peripheral blood.PCR was performed to amplify each exon and the surrounding parts of theinrons of EPB42genes. The PCR products were screened by direct sequencing.ResultsThere was not shown prominent changes in membrane protein levels ofSDS-PAGE results. Relative mRNA quantification of the EPB42transcript leveldecreased in HS patients when compared to the control group Two EPB42genepolymorphisms were identified by PCR and subjected to direct sequencing, c.329C>T and c.720G>A. The SNP (c.329C>T) was detected in the patient’smother.Conclusion1. We have established the diagnostic platform of relative mRNAquantification of the EPB42gene using real-time PCR, to select defects oferythrocyte membrane proteins.2. Real-time PCR experiments indicated that the4.2protein gene (EPB42)could be involved in anaemia pathogenesis.3. EPB42gene changes, c.329C>T, may be related to HS.