Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel.
PLoS One. 2010 Nov 16;5(11):e13659. doi: 10.1371/journal.pone.0013659.
Leukocyte adhesion deficiency 1 (LAD1) is an inherited disorder of neutrophil function. Nonsense mutations in the affected CD18 (ITB2) gene have rarely been described. In other genes containing such mutations, treatments with aminoglycoside types of antibiotics (e.g., gentamicin) were reported to partially correct the premature protein termination, by induction of readthrough mechanism.
METHODOLOGY/PRINCIPAL FINDINGS: Genetic analysis was performed on 2 LAD1 patients. Expression, functional and immunofluorescence assays of CD18 in the patients were used to determine the in-vivo and in-vitro effects of gentamicin-induced readthrough. A theoretical modeling of the corrected CD18 protein was developed to predict the protein function.
We found a novel premature termination codon, C562T (R188X), in exon 6 of the CD18 gene that caused a severe LAD1 phenotype in two unrelated Palestinian children. In-vivo studies on these patients' cells after gentamicin treatment showed abnormal adhesion and chemotactic functions, while in-vitro studies showed mislocalization of the corrected protein to the cytoplasm and not to the cell surface. A theoretical modeling of the corrected CD18 protein suggested that the replacement of the wild type arginine by gentamicin induced tryptophan at the position of the nonsense mutation, although enabled the expression of the entire CD18 protein, this was not sufficient to stabilize the CD18/11 heterodimer at the cell surface.
A novel nonsense mutation in the CD18 gene causing a complete absence of CD18 protein and severe LAD1 clinical phenotype is reported. Both in vivo and in vitro treatments with gentamicin resulted in the expression of a corrected full-length dysfunctional or mislocalized CD18 protein. However, while the use of gentamicin increased the expression of CD18, it did not improve leukocyte adhesion and chemotaxis. Moreover, the integrity of the CD18/CD11 complex at the cell surface was impaired, due to abnormal CD18 protein and possibly lack of CD11a expression.
白细胞黏附缺陷 1（LAD1）是一种中性粒细胞功能遗传性疾病。在受影响的 CD18（ITB2）基因中，罕见有报道无义突变。在其他含有此类突变的基因中，氨基糖苷类抗生素（如庆大霉素）的治疗被报道通过诱导通读机制部分纠正过早的蛋白终止。
方法/主要发现：对 2 例 LAD1 患者进行了基因分析。通过患者 CD18 的表达、功能和免疫荧光检测，确定庆大霉素诱导通读的体内和体外作用。开发了一种校正后 CD18 蛋白的理论模型，以预测蛋白功能。
我们在 CD18 基因的外显子 6 中发现了一个新的提前终止密码子 C562T（R188X），导致两名无关联的巴勒斯坦儿童出现严重的 LAD1 表型。庆大霉素治疗后这些患者细胞的体内研究显示异常黏附和趋化功能，而体外研究显示校正蛋白错误定位到细胞质而不是细胞表面。校正后 CD18 蛋白的理论模型表明，尽管庆大霉素诱导无义突变处的色氨酸取代野生型精氨酸，从而使整个 CD18 蛋白得以表达，但这不足以稳定细胞表面的 CD18/11 异二聚体。
报告了一种新的 CD18 基因突变，导致完全缺乏 CD18 蛋白和严重的 LAD1 临床表型。体内和体外用庆大霉素治疗均导致表达校正的全长功能失调或定位错误的 CD18 蛋白。然而，尽管庆大霉素增加了 CD18 的表达，但并未改善白细胞黏附和趋化作用。此外，由于 CD18 蛋白异常和可能缺乏 CD11a 表达，细胞表面的 CD18/CD11 复合物完整性受损。
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