摘要目的:分析SPTB基因新的内含子变异并探讨该变异对SPTB基因mRNA剪接的影响。方法:回顾性总结西安交通大学第一附属医院2022年2月收治的1例遗传性球形红细胞增多症（HS）患儿及其家系的临床资料，采用全基因组测序检测致病变异并进行Sanger测序验证，mRNA测序分析SPTB基因mRNA表达水平分析，利用生物信息学工具对剪接位点进行预测与分析。结果:先证者为2月龄汉族男性患儿，临床表现为贫血、黄疸，既往新生儿期黄疸出现早、程度重，间接胆红素显著升高（203.5 μmol/L），同时伴中度贫血。患儿家系共4代，其中8例有贫血、黄疸、脾大等临床表现，外周血球形红细胞增多，占5%~10%；其中先证者父亲外周血红细胞扫描电镜下口形红细胞约占红细胞总数的6%、球形红细胞约占4%、椭圆形红细胞约占3%，经脾切除治疗后贫血及黄疸均恢复正常。先证者全基因组测序分析发现SPTB基因的1个杂合变异［NM_ 001355436.2（SPTB）：c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC］，经Sanger测序验证该变异和疾病在此家系中共分离。SPTB基因mRNA表达水平检测结果显示致病基因SPTB在先证者及家系成员中患者（父亲、祖母、表姨、表姑、曾祖母、大姨祖母）的表达水平均高于对照样本，差异均有统计学意义（均 P<0.05）。利用生物信息软件ESE Finder分析发现SPTB基因内含子变异导致mRNA选择性剪接，SpliceAI及SpliceTool软件预测均显示新的隐蔽剪接供体激活，导致移码变异，引入提前终止密码子并出现无义介导的mRNA降解，从而阻断蛋白的合成。 结论:本研究发现了SPTB基因新的变异位点c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC，该变异是本家系HS的致病原因。该变异通过影响剪切过程触发无义介导的mRNA降解途径，从而导致基因功能失活。

abstractsObjective:To analyze a novel intronic variant in the SPTB gene and explore its effect on SPTB mRNA splicing.Methods:Clinical data of a child diagnosed with hereditary spherocytosis (HS) and admitted to the First Affiliated Hospital of Xi′an Jiaotong University in February 2022 were analyzed retrospectively. Whole genome sequencing was used to identify disease-causing variantions and the results were validated with Sanger sequencing, mRNA sequencing was used to determine the SPTB gene′s mRNA expression level, and bioinformatics tools were used for splicing site prediction and analysis.Results:The proband is a 2-month-old Han male child, clinically presenting with anemia and jaundice. In the past, jaundice appeared early and was severe during the neonatal period, with significantly elevated indirect bilirubin (203.5 μmol/L), accompanied by moderate anemia. This family consisted of four generations, eight of whom suffered from splenomegaly, jaundice, and anemia. In their peripheral blood, the percentage of microglobular erythrocytes was between 5% and 10%. Under scanning electron microscopy analysis of the proband's father's peripheral red blood cells, about 6% exhibited a mouth-shaped morphology, about 4% were spherical, and about 3% were oval. Following the splenectomy, the father′s anemia and jaundice recovered to normal level. Whole genome sequencing analysis of the proband identified a heterozygous variant in the SPTB gene (NM_ 001355436.2 (SPTB):c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC), which was verified to be co-segregating with the disease in this family line by Sanger sequencing. The results of the SPTB gene mRNA expression level detection showed that the expression levels of the SPTB variant gene were statistically increased in the proband and affected family members (father, grandmother, cousin, second cousin, great-grandmother, great-aunt) (all P<0.05). The SPTB gene′s intron can undergo selective splicing, as demonstrated by analysis using the bioinformatics program ESE Finder. Additionally, predictions from the SpliceAI and SpliceTool software indicated that activation of a new covert splicing donor can result in a code-shift variantion that introduces an early termination codon and nonsense-mediated degradation of the mRNA, which prevents the synthesis of proteins. Conclusion:A new variantion site c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC was found in SPTB gene. This variantion was the pathogenic factor of HS. By affecting the splicing process, this variantion triggers the nonsense mediated mRNA degradation pathway, resulting in inactivation of gene function.