不同类型WASP基因突变对湿疹、血小板减少伴免疫缺陷综合征造血干细胞体内生长的影响

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disease caused by mutations in the Wiskott-Aldrich Protein (WASP) gene, which typically leads to absent WASP protein (WASp) expression in WAS leukocytes. However, some patients have been found with small populations of WASP-expressing cells caused by reverse or second-site mutations that allow protein expression. Reversion appears to be a phenomenon of relatively high incidence in WAS patients and were noted in approximately 10% of WAS patients tested. It had resulted in progressively improving or mild clinical phenotype in some patients with reversions. But the mechanisms of revision of WASP gene is not clear up to now. Selective advantage of revertant cells in vivo perhapes is one of the mechanisms. The enrichment of revertant cells might have been caused by the selective advantage of WASP+ compared with WASP- progenitor or precursor cells in migratory capacity, chemokine responsiveness, proliferative responsiveness, or survival capacity. In the study, SCID mice were transplanted with human bone marrow derived CD34+ cells from five WAS patients with different mutations of WASP gene after conditioning by irradiation, including missense mutation, nonsense mutation, insertion mutation, deletion mutation and splice-site mutation. CD34+ cells from XLT patient with WASp expression were used as control and mixed with CD34+ cells from WAS patients with different mutations before transplantation. At four, eight, twelve weeks post-reconstitution, mice were screened for human cell engraftment in peripheral blood, spleen, bone marrow by staining for human leukocytes marker followed by FACS analysis and molecular biological methods. WASp- and WASp+ leukocytes cells were regarded as from CD34+ cells of WAS and XLT patients respectively. Tissue sections of lymphoid organs such as spleen, lymph node, and thymus from SCID mice were stained for the presence of human leukocytes and WASp. It will elucidate the selective advantage of development and immune function after transplantation of bone marrow hematopoietic stem cells with different mutations of WASP gene and help strategy selection of stem cell transplantation and gene therapy.

回复突变是湿疹、血小板减少伴免疫缺陷综合征（WAS）较常见的自然遗传学现象，但致病基因完全恢复至正常或相对正常的机制尚未阐明。回复突变的细胞在体内具有生长优势可能是致病机制之一。本研究拟在前期工作基础上，主要采用流式细胞术及分子生物学方法，利用严重联合免疫缺陷（SICD）小鼠动物模型，进一步阐明不同类型WASP基因突变的骨髓造血干细胞的体内竞争生长和免疫功能变化规律，验证回复突变的"生长优势"假说。本研究以稀有临床资源为基础，揭示的回复突变机制有助于本病造血干细胞移植和基因治疗策略制定。

湿疹、血小板减少伴免疫缺陷综合征是一种复杂且严重的X连锁隐性遗传性疾病。研究周期内我们完成了国内最大样本的WAS基因型和表现型关系的研究，结果提示WAS基因突变与WAS患儿临床表型及远期预后有潜在关联，而环境因素的影响也不容忽视。错义突变患儿可有一定水平WASp表达，临床表型较轻，远期预后相对较好。典型WAS患儿应及早接受免疫重建治疗。进一步我们发现5例回复突变的WAS患儿，并动态随访其临床特征和重要免疫细胞功能的变化。结果提示回复突变对临床表型的改善不全面。感染和湿疹有所改善，存活时间延长，但血小板减少症和自身免疫性疾病发生并未减轻。本研究还发现回复突变主要发生在T细胞，且回复突变细胞比例有进行性增多趋势，可能与感染等表现的改善有关。但这种生长优势是否会导致临床表现持续向好，免疫功能进一步恢复尚不清楚，还有待进一步研究。此外，我们还发现在WAS病例中，自身免疫性疾病发生率较高，以自身免疫性溶血性贫血为主。合并自身免疫性疾病的WAS患儿应尽早行造血干细胞移植。

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