Speletas M, Kanariou M, Kanakoudi-Tsakalidou F, Papadopoulou-Alataki E, Arvanitidis K, Pardali E, Constantopoulos A, Kartalis G, Vihinen M, Sideras P, Ritis K
First Division of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece.
Scand J Immunol. 2001 Sep;54(3):321-7. doi: 10.1046/j.1365-3083.2001.00967.x.
Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase, critical for B-cell development and function. Mutations that inactivate this kinase were found in families with X-linked agammaglobulinaemia (XLA). In this study the Btk gene was analyzed in 13 registered Greek patients with XLA phenotype originated from 12 unrelated families, in order to provide a definite diagnosis of the XLA. The structure of Btk was analyzed at the cDNA level using the recently developed method, NIRCA (Non-Isotopic-Rnase-Cleavage-Assay). Alterations were detected in all patients and sequencing analysis confirmed the results and defined six novel XLA-associated Btk mutations (three missense mutations: C337G, L346R, L452P; one nonsense mutation: Y392X, and two frameshift alterations: c1211-1212delA, c1306-1307insA). Having defined the genetic alteration in the affected males of these families, the information was used to design polymerase chain reaction (PCR) primers and the Btk segments containing the mutated sequences were amplified from peripheral blood derived genomic DNA of potential female carriers. The PCR products were directly sequenced and carrier status was determined in 12 out of 16 phenotypically normal females analyzed. This protocol can be used once the nature of the Btk mutation has been defined in one of the affected males and provides a convenient, simple and reliable way to determine the carrier status of other female family members. Molecular genetic analysis constitutes a determinative tool for the definitive diagnosis of XLA and may allow accurate carrier and prenatal diagnosis for genetic counselling.
布鲁顿酪氨酸激酶（Btk）是一种非受体酪氨酸激酶，对B细胞的发育和功能至关重要。在患有X连锁无丙种球蛋白血症（XLA）的家族中发现了使该激酶失活的突变。在本研究中，对来自12个无关家族的13名登记在册的希腊XLA表型患者的Btk基因进行了分析，以便对XLA做出明确诊断。使用最近开发的方法——非同位素核糖核酸酶切割分析（NIRCA），在cDNA水平分析Btk的结构。在所有患者中均检测到改变，测序分析证实了结果，并确定了六个新的与XLA相关的Btk突变（三个错义突变：C337G、L346R、L452P；一个无义突变：Y392X，以及两个移码改变：c1211 - 1212delA、c1306 - 1307insA）。确定了这些家族中受影响男性的基因改变后，利用该信息设计聚合酶链反应（PCR）引物，并从潜在女性携带者的外周血基因组DNA中扩增包含突变序列的Btk片段。对PCR产物进行直接测序，在分析的16名表型正常的女性中，有12名确定了携带者状态。一旦在一名受影响男性中确定了Btk突变的性质，就可以使用该方案，它提供了一种方便、简单且可靠的方法来确定其他女性家庭成员的携带者状态。分子遗传学分析是XLA明确诊断的决定性工具，并且可能有助于进行准确的携带者诊断和产前诊断以进行遗传咨询。
