SLC4A1 Matations In Chinese Pedigrees With Hereditary Spherocytosis

Objective:Hereditary spherocytosis (HS) is a common inherited hemolytic disease characterized by anaemia, jaundice, and splenomegaly as well as the presence of spherical-shaped erythrocytes on the peripheral blood smear. HS has a heterogeneous clinical presentation, molecular basis, and inheritance. HS is caused by mutations in genes encoding red blood cell membrane proteins. Mutations in five genes, ANK1, SLC4A1, SPTA1, SPTB, and EPB42, which encode ankyrin, band 3, α-spectrin,β-spectrin, and protein 4.2, respectively, have previously been shown to be causative of HS. SLC4A1 gene mutations include, but are not limited to, missense, nonsense, insertions, and deletions for Europe and America. The molecular pathological mechanism underlying HS has been studied in patients from Europe and America, but few investigations have been made of Chinese HS patients.The identification of defects in red blood cell membrane proteins by gel electrophoresis (SDS-PAGE and Western Blot) and genomic DNA sequencing are two common methods for studying HS. However, SDS-PAGE and Western Blot are fairly insensitive to red cell membrane proteins. In the present study, therefore, we first used SDS-PAGE and Western Blot to identify the type of red blood cell membrane protein deficiency in a Chinese pedigree with HS, and then analyzed all exons and adjacent introns of SLC4A1 using genomic DNA sequencing. We validated these mutation sites in family members to investigate the pathological mechanism underlying HS patients in China, and to benefit genetic counseling services.Method:In this study,we collected peripheral blood from five HS patients related to band 3 deficiency and their family members; using traditional method extracted erythrocyte membrane protein;using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot technology analyzed the type of erythrocyte membrane protein defect. According to DNA kit instructions, peripheral blood DNA was extracted from HS patients; The corresponding primers were designed and synthesized for band 3 defect(SLC4A1 gene),and obtain the target products by PCR; using genomic DNA sequencing technology analyze all exons and adjacent introns of SLC4A1 gene, and compared with the standard sequence in the NCBI database and analyze and verify gene mutation form. Then we validated these mutation sites in family members, known gene mutation form of the SLC4A1 gene with HS, and investigated the pathological mechanism underlying HS patients related to band 3 deficiency in China.ResultThe probands of five families are all band 3 deficiency by SDS-PAGE and Western Blot. Mutations were detected in SLC4Algene by genomic DNA sequencing:Proband 1 was band 3 partial deficiency, two mutations were detected in SLC4A1 gene:substitution Aâ†'C in nucleotide 113, changing codon 38 from GAC to GCC (Aspâ†'Ala), a missen mutation in exon 4; and a single base substitution in the splicing donor site of intron 6(position +27Câ†'T) are identified.Proband 2 was band 3 partial deficiency, two mutations were detected in SLC4A1 gene:substitution Câ†'T in nucleotide 1540, changing codon 514 from CGC to TGC (Cysâ†'Arg), a missen mutation in exon 13; and a single base substitution in the splicing donor site of intron 7(position+86Gâ†'A) are identified.Proband 3 was band 3 partial deficiency, two mutations were detected in SLC4A1 gene:substitution Aâ†'C in nucleotide 113, changing codon 38 from GAC to GCC (Aspâ†'Ala), a missen mutation in exon 4; and a single base substitution in the splicing donor site of intron 6(position+27Câ†' T) are identified.Proband 4 was band 3 partial deficiency, two mutations were detected in SLC4A1 gene:substitution Aâ†'G in nucleotide 166, changing codon 56 from GAC to GCC (Lysâ†'Glu), a missen mutation in exon 4;and substitution Aâ†'C in nucleotide 113, changing codon 38 from AAG to GAG (Aspâ†'Ala), a missen mutation in exon 4 are identified.Proband 5 was band 3 partial deficiency, one mutations was detected in SLC4A1 gene:substitution Aâ†'G in nucleotide 166, changing codon 56 from AAG to GAG (Lysâ†'Glu), a missen mutation in exon 4.Conclusion1. It is successful that applying Western Blot to explore the type of erythrocyte membrane protein defect with HS patients. Western Blot and SDS-PAGE Coomassie Blue Staining of both methods can detect membrane protein defect types more specifically.2. HS patients gene mutations scattered distribution, even though the same protein defect, HS patients with different pedigrees almost have its own type of gene mutation. However, HS patients with a pedigree showed gene mutations in the same way.3. This study shows that the majority of the Chinese population of patients with HS oriented focus on band 3 deficiency.