Table1_Effect of primary lesions in cytoskeleton proteins on red cell membrane stability in patients with hereditary spherocytosis.DOC

We investigated by targeted next generation sequencing the genetic bases of hereditary spherocytosis in 25 patients and compared the molecular results with the biochemical lesion of RBC membrane obtained by SDS-PAGE analysis. The HS diagnosis was based on available guidelines for diagnosis of congenital hemolytic anemia, and patients were selected because of atypical clinical presentation or intra-family variability, or because presented discrepancies between laboratory investigation and biochemical findings. In all patients but 5 we identified pathogenic variants in SPTA1, SPTB, ANK1, SLC4A1, EPB42 genes able to justify the clinical phenotype. Interestingly, a correspondence between the biochemical lesion and the molecular defect was identified in only 11/25 cases, mostly with band 3 deficiency due to SLC4A1 mutations. Most of the mutations in SPTB and ANK1 gene didn’t hesitate in abnormalities of RBC membrane protein; conversely, in two cases the molecular lesion didn’t correspond to the biochemical defect, suggesting that a mutation in a specific cytoskeleton protein may result in a more complex RBC membrane damage or suffering. Finally, in two cases the HS diagnosis was maintained despite absence of both protein defect and molecular lesion, basing on clinical and family history, and on presence of clear laboratory markers of HS. The study revealed complex relationships between the primary molecular lesion and the final effect in the RBC membrane cytoskeleton, and further underlines the concept that there is not a unique approach to the diagnosis of HS.

本研究采用靶向下一代测序（targeted next generation sequencing）技术，对25例遗传性球形红细胞增多症（hereditary spherocytosis, HS）患者的致病遗传基础进行探究，并将分子检测结果与通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析得到的红细胞（red blood cell, RBC）膜生化损伤结果进行对比。本研究的HS诊断参照先天性溶血性贫血诊断的现行临床指南，入组患者均为临床表现不典型、存在家族内表型异质性，或实验室检测结果与生化检测结果不符的病例。除5例患者外，其余所有病例均在SPTA1、SPTB、ANK1、SLC4A1、EPB42基因中检出可合理解释临床表型的致病性变异。值得注意的是，仅11/25例病例的生化损伤与分子缺陷存在对应关系，此类病例多为SLC4A1突变引发的带3蛋白（band 3 protein）缺乏症。多数SPTB与ANK1基因的突变并未引发红细胞膜蛋白异常；与之相反，有2例病例的分子缺陷与生化损伤并不匹配，这提示特定细胞骨架蛋白的突变可能引发更为复杂的红细胞膜损伤或功能异常。最终，有2例病例虽未检出蛋白缺陷与分子病变，但结合临床与家族病史，以及明确的HS实验室标志物，仍维持HS诊断。本研究揭示了原发性分子病变与红细胞膜细胞骨架最终表型之间的复杂关联，并进一步强调：HS的诊断并无统一的单一方案。