Pienaar S, Eley B, Beatty D W, Henderson H E
Institute of Child Health, University of Cape Town, Red Cross Children's Hospital, South Africa.
J Paediatr Child Health. 2000 Oct;36(5):453-6. doi: 10.1046/j.1440-1754.2000.00546.x.
Molecular analysis of the Bruton's tyrosine kinase (Btk) gene in two unrelated families, with a combined total of seven boys, affected by X-linked agammaglobulinaemia (XLA).
Protein electrophoresis and western blotting were used for the examination of Btk protein synthesis in blood leucocytes. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction (RT-PCR) technique. Sequence alterations were screened for by the single-stranded conformation polymorphism (SSCP) method and characterized by standard sequencing protocols.
Western blotting revealed Btk protein to be absent in leucocytes of affected males from both families. A novel 3 b.p. deletion in exon 3 of the Btk gene was found to be responsible for the XLA phenotype in the affected proband in one family (kindred I). A diagnostic PCR assay was established to detect this mutation in other affected male siblings and carrier females. For the second family (kindred II), the coding sequence of the Btk gene and the promoter region were found to be normal.
The present study has demonstrated genetic heterogeneity in the Btk gene in South African XLA patients and has identified a novel mutation in this gene in the largest of the affected kindreds. The gene mutation in the second kindred was undetermined and may be indicative of a defect in some other gene associated with Btk function or stability. Western blotting was found to be informative in establishing a deficiency of Btk protein in both probands and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.
对两个无血缘关系的家庭中总共七名患X连锁无丙种球蛋白血症（XLA）的男孩进行布鲁顿酪氨酸激酶（Btk）基因的分子分析。
采用蛋白质电泳和蛋白质印迹法检测血白细胞中Btk蛋白的合成情况。通过逆转录-聚合酶链反应（RT-PCR）技术扩增来分离Btk基因的编码序列。采用单链构象多态性（SSCP）方法筛选序列改变，并通过标准测序方案进行特征分析。
蛋白质印迹显示两个家庭中患病男性的白细胞均不存在Btk蛋白。在一个家庭（家系I）中，发现Btk基因外显子3中有一个新的3个碱基对的缺失，这导致了患病先证者出现XLA表型。建立了诊断性PCR检测方法，以检测其他患病男性同胞和携带者女性中的这种突变。对于第二个家庭（家系II），发现Btk基因的编码序列和启动子区域正常。
本研究证明了南非XLA患者Btk基因存在遗传异质性，并在最大的患病家系中鉴定出该基因的一种新突变。第二个家系中的基因突变未确定，可能表明与Btk功能或稳定性相关的其他基因存在缺陷。发现蛋白质印迹对于确定两名先证者中Btk蛋白缺乏情况具有参考价值，建议将其作为XLA分子诊断和检查的一线方法。
