| ObjectiveThis study aimed to summarize the clinical phenotypic features of children with anemia that cannot be identified by routine clinical diagnosis,distinguish the clinical characteristics of such children earlier,and explore genetic screening strategies for childhood hereditary spherocytosis(HS).The genetic background of HS was studied through genetic testing and pathogenicity assessment to clarify the main genetic variation types and frequency distribution of HS in children,further enriching the mutation spectrum of HS.The functions of the mutated gene loci were investigated in vitro to explore HS pathogenesis preliminarily.The clinical characteristics of children with HS were collected,and the association between different genotypes and phenotypes of the disease was clarified.Method1)Children with anemia that routine methods could not determine were included in this study.Clinical data were collected,including age,gender,blood routine,liver and spleen size,family history,and splenectomy.Peripheral venous blood 4 m L(EDTA anticoagulation)and 2 m L of parental venous blood were collected for whole-exome sequencing and Sanger sequencing.2)The sequencing results of 19 children with HS and the previously published ANK1 and SPTB gene information in NCBI retrieved by September 2021 were summarized and analyzed.3)A minigene test was performed on the mutated sites near the splicing site(ANK1.1504-9(IVS 13)G>A).4)The clinical data of 19 children with HS confirmed by genetic testing were collected.Children with autoimmune hemolytic anemia were admitted to our hospital during the same period.Other children with positive/negative genes in Part I of the study were included as controls.Patients were grouped according to disease type/different mutant gene/mutant type/mutant domain.Hb,RBC,HCT,MCV,MCH,MCHC,RDW-SD,RDW-CV,RET,PLT,TBIL,DBIL,and IDBL were counted.Statistical analysis was performed using the Mann-Whitney test between groups,and p<0.05 was considered a significant difference.Results1)In this study,30 children with anemia of unknown causes were enrolled.The male-to-female ratio was 1:1.5,and the median age was 3 years and 10 months.The total positive rate of gene detection was 83.33%(25/30),and the non-HS-related congenital anemia genes were 20%(6/30),including GPI,PKLR,GATA1,CUBN,HBB,and CFHR1.Nineteen cases were identified as HS(63.33%,19/30),including12 cases with ANK1 mutation(63.15%,12/19)and 7 cases with SPTB mutation(36.84%,7/19).2)Among the 19 HS pathogenic genes,13 were de novo,4 maternal inheritance,and 2 paternal inheritance.There were 18 different mutation sites,including 10 nonsense mutations,5 frameshift mutations,and 3 splice sites or nearby mutations,and 12 mutation sites were reported for the first time in our study.3)Tested by minigene in vitro experiments,the mutation of ANK1 c.1504-9(IVS 13)G>A could affect the normal splicing of m RNA.The detection results of the two sets of vectors consistently showed that a new splice acceptor site was generated at the mutation,resulting in a 7 nt retention at the 5’ end of intron 13,further verifying the pathogenicity of this site.4)The median age of the 19 HS patients was 3 years and 6 months,and the male-to-female ratio was 7:12.Splenomegaly and gallstones were found in 11 patients.Six other children with congenital anemia were aged between 1 month and10 days to 12 years and 1 month.The male-to-female ratio was 2:1,including one case of splenomegaly.Five patients with unknown etiology were from 2 years and 5months to 11 years and 4 months,and the male-to-female ratio was 1:4,with 3 cases of splenomegaly.The median age of the 15 children with AIHA was 2 years and 10 months,and the male-to-female ratio was 8:7.Splenomegaly/gallstones were not obvious during the initial diagnoses.5)We performed an intergroup comparison of 19 HS patients and 6 other congenital anemia patients.The RDW-CV and RDW-SD of children with HS were significantly higher than those of other children with congenital anemia(p=0.0255,p=0.0135,respectively).There were no significant differences in the other indicators between the two groups(p>0.05).6)We compared 19 children with HS and 5 children with gene-negative anemia of unknown causes.The RBC count of children with HS was lower than that of the control group(p=0.0487),and the RET count was higher than that of the control group(p=0.0365).There were no significant differences in the other indicators between the two groups(p>0.05).7)We also compared 19 HS patients with 15 AIHA patients between groups.RBC(p=0.0095)and HB(p=0.0157)in children with HS were significantly higher than those in children with AIHA,and MCV(p=0.0010),MCH(p=0.0023)were substantially lower in the HS group than in the AIHA group.There were no significant differences in the other indicators between the two groups(p>0.05).8)In our study,19 children with HS were grouped and compared according to nonsense and frameshift mutations,there was no significant statistical difference between the two groups(p>0.05).9)We further divided 19 HS children into an ANK1 group and SPTB group according to different pathogenic genes for inter-group comparison.We found that RBC(p=0.0073),HB(p=0.0054)and HCT(p=0.0107)levels were significantly lower in the ANK1 group than in the SPTB group.There were no significant differences in the other indicators between the two groups(p >0.05).10)Finally,children with HS who carried alleles were divided into groups according to the difference in the lost domain.The indices of Hb,RBC,MCV,MCH,MCHC,and other indicators were compared again.Still,the number was too small to be statistically significant.Conclusion1)The positive rate of HS genetic screening can be increased by secondgeneration sequencing after excluding the conventional causes of anemia.2)NGS can be used to determine the probable cause of previously undiagnosed cases of anemia when the clinical phenotype is insufficient to determine the cause of anemia.3)We reported 12 new mutation sites for the first time,further enriching the spectrum of the HS pathogenic gene mutation sites.4)We clarified the mutation characteristics of major gene deletion types and distribution frequency in children with HS: the ANK1 gene mutation is the most prominent cause of HS,followed by the SPTB gene.The main types of mutations are nonsense and frameshift mutations.5)ANK1 c.1504-9(IVS13)G>A mutation disturbed the normal splicing of gene m RNA,preliminarily verifying the pathogenesis.6)The clinical characteristics of HS at initial diagnosis are not specific.When the clinical diagnosis of HS is unknown,second-generation sequencing remains the main diagnostic and differential diagnosis method.7)For the first time,we analyzed the association between different genotypes and phenotypes of HS without the influence of splenectomy factors on HS indicators,ANK1-HS was more severe in anemia than SPTB-HS. |
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