Study No The Erythrocyte Membrance Protein Defect Types And Mutation Spectra Of The 5 Families Of Hereditary Spherocytosis

Background and ObjectiveHereditary spherocytosis (HS) is a common inherited disorder that is characterised by anaemia, jaundice, and splenomegaly. It is reported worldwide and in northern Europe and North America where it affects one person in 2000. Approximately 75% of HS showed autosomal dominant inheritance, about 25% of the HS is manifested as an autosomal inherited or de novo mutation invisible. Mutations affecting the genes SPTA1, SPTB, ANK1, SLC4A1 and EPB42, encoding for the erythrocyte membrane proteins spectrin α-chain,spectrin, β-chain, ankyrin, the anion exchanger 1 (band 3) and protein 4.2, respectively. Once one or more of α-spectrin, β-spectrin, ankyrin, band-3 protein and protein 4.2 defect can cause red vertical connection between the lipid bilayer membrane skeleton and weakened, then double-concave disc-shaped red blood cells become spherical red blood cells with the cell membrane to the microcapsules bubble in the form of lost.Diagnosis of HS is currently based on a combination of clinical, the patient’s family history and laboratory tests. Paula HB Bolton-Maggs et al. pointed a patient with a family history of HS, typical clinical features and laboratory investigations (spherocytes, raised mean corpuscular haemoglobin concentration [MCHC], increase in reticulocytes) do not require any additional tests can diagnosing HS in the guidelines for the diagnosis and management of hereditary spherocytosis which published in 2011. In practical work, however, a considerable part of HS patients are not have all of the characteristics above, they are often misdiagnosed. SDS-PAGE is classical diagnostic method of HS which can determine the HS patient membrane protein defects. However, SDS-PAGE lacks sensitivity to very mild or asymptomatic HS carriers. Mariagabriella Mariani analysed a large database of 300 patients with hereditary spherocytosis grouped according to the results of SDS-PAGE, they found that 11% of cases were not classified by SDS-PAGE analysis.With the rapid development of genetic diagnostic techniques, it brings a new direction for HS. Nowdays, it has been found missense, insertions and deletions, and other types in the presence of point mutations. However, the hot spot has not yet been found in HS mutations. In China, the studies of HS are limited case reports, that few studies on the HS gene mutation detection.MethodIn this study, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) used to identify the erythrocyte membrane proteins defect causing HS; Peripheral blood DNA extracted from patients, the corresponding primers were designed and synthesized,amplified by PCR to obtain the target product. Used direct DNA sequencing method, compared with the standard sequence within the NCBI database, to proven gene mutation form. Molecular genetic analyses of the patient and the parents were performed.ResultsRed cell protein 4.2 was completely missing in the proband 1, and nucleotide sequence analysis of the EPB42 exon 3 revealed a single base substitution at g.5935 G>A heterozygous state. Band-3 protein partial defects in the proband 2, a heterozygous mutation g.6505 C>A in exon 4 and g.6557 T>C in intron 4 of the SLC4A1 gene was detected in the patient. Band-3 protein partial defects in the proband 2, a heterozygous mutation g.6505 C>A in exon 4 of the SLC4A1 gene was detected in the patient. A heterozygous mutation g.55554 A>T in exon 29 of the SPTB gene was detected, in the proband 5, without no obvious defects proteins. A heterozygous mutation g.7367 T>G in exon 5 of the SLC4A1 gene was detected in the patient with band-3 protein partial defects in the proband 5.Conclusion1. SDS-PAGE have a good diagnostic value for the HS patients with erythrocyte membrane protein completely deficient, but lacks sensitivity to the part of the erythrocyte membrane protein defects.2. The emphasis on family research and application MSCV can improve the diagnostic rate of HS.3. HS patients’mutation sites scattered distribution, which HS patients with a pedigree showed the same mutation way.