Molecular Genetic Study Of Pathogenic Genes In Two Families With Hereditary Spherocytosis

Objective:To identify the pathogenic genes,pathogenic variants,and possible molecular pathogenic mechanisms in two families with hereditary spherocytosis（HS）,to provide a theoretical basis for screening other asymptomatic variant carriers in the families for appropriate life or medical management in advance and enrich the variant spectrum of HS-related pathogenic genes in the Chinese population.Methods:In the present study,two HS families from Gansu Province were recruited.Clinical data and peripheral blood samples of patients were collected.First,whole-exome sequencing was performed on the peripheral blood DNA of the proband to identify suspicious pathogenic genes and variant sites.Then,the suspected pathogenic variants were verified among the proband and other members of the families by Sanger sequencing.Finally,the molecular genetics of the variant sites was studied by RT-PCR,q PCR,Western-blot,immunofluorescence,and co-immunoprecipitation techniques to explore its possible pathogenic mechanism.In addition,the content of reactive oxygen species（ROS）in erythrocytes and the expression of Nrf2,CAT,and SOD genes in HS patients were detected to further determine the existence of oxidative stress in patients with HS and to explore whether the antioxidant stress signal pathway Nrf2-Keap1/ARE was dysfunctional in HS patients.Results:In this study,a novel pathogenic heterozygous frameshift variant（c.1509＿1518del GGACAATATA,p.K503Nfs*67）in the SPTB gene was identified in pedigree 1 HS patients,which disrupted the synthesis and localization ofβⅠspectrin and weakened the interaction betweenβⅠspectrin and ankyrin,possibly due to a nonsense-mediated m RNA degradation pathway.A known pathogenic heterozygous nonsense variant（c.5266C>T,p.Arg1756*）in the SPTB gene was found in patients with HS of pedigree 2,and we further perfected the pathogenic mechanism of this variant.The variant produced a truncatedβⅠspectrin,which may be integrated into the full-lengthβⅠspectrin,leading to changes in the structure of theα₂β₂spectrin tetramerization.In addition,we found that the content of ROS in red blood cells of patients with HS was increased,and the expression levels of Nrf2,CAT,and SOD genes in peripheral blood were decreased,indicating that there was oxidative stress in patients with HS and the Nrf2-keap1/ARE signal pathway was dysfunctional.Conclusion:This study found two pathogenic variants related to HS in the SPTB gene and elaborated the possible molecular pathogenic mechanism,which expanded the spectrum of SPTB variants,especially in the Chinese population,and increased the current understanding of the molecular mechanisms related to premature termination codon（PTC）variants in the SPTB gene.