Identification and functional characterization of a novel heterozygous splice-donor (c.647+1G>A) site mutation in SPTB gene causes hereditary spherocytosis with hemolytic anemia

KE CAO¹Xiaojuan Luo¹Lianlian Liu¹Xiaoning Mao¹Ruping Liu²Yunsheng Chen¹Prof. (Dr.) Santasree Banerjee^2*

• ¹Shenzhen Children's Hospital, Shenzhen, China
• ²Jilin University, Changchun, China

Objective: Hereditary spherocytosis (HS) is an inherited disorder characterized by spherical-shaped erythrocytes and abnormalities of several erythrocyte membrane proteins with extreme genotypic and phenotypic heterogeneity. HS patients were clinically diagnosed by the presence of spherical-shaped erythrocytes on the peripheral blood smear, haemolytic anaemia, jaundice, splenomegaly, with or without cholelithiasis or gallstones. Till date, mutations of five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) have been reported to be associated with different subtypes of HS. Germline mutations of SPTB gene causes autosomal dominant HS (Spherocytosis 2, SPH2), the rarest subtypes of HS. Methods: In this study, we investigated a 10-year-old Chinese girl clinically diagnosed with HS and neonatal hemolytic anemia. Proband's mother was also identified with HS and hemolytic anemia but proband's father was phenotypically normal. We performed standard G-banding karyotype to identify structural abnormalities of chromosomes in this proband. Then, we performed whole exome sequencing and Sanger sequencing to identify the disease-causing variants in this proband. Finally, we functionally characterize the identified novel variant by performing reverse transcription polymerase chain reaction, cDNA sequencing, quantitative real time PCR and western blot. Results: Whole exome sequencing identified a heterozygous novel splice-donor-site (c.647+1G>A) mutation in SPTB gene in the proband. Sanger sequencing confirmed that the proband was inherited this mutation from her mother, while her father was devoid of it. Reverse transcription polymerase chain reaction and cDNA sequencing showed that this novel splice-donor-site (c.647+1G>A) mutation causes abolition of wild type splice donor site which leads to the aberrant splicing of SPTB mRNA followed by the formation of alternative transcript with complete loss of exon 5. Relative expression of mutated SPTB mRNA was significantly reduced in the proband and her mother compared with her father showing normal expression of wild type SPTB mRNA. Conclusion: Our present study highlighted the significance of whole exome sequencing as the most potential way for genetic molecular diagnosis for the patients with HS.