Analysis of Btk mutations in patients with X-linked agammaglobulinaemia (XLA) and determination of carrier status in normal female relatives: a nationwide study of Btk deficiency in Greece

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase, critical f or B-cell development and function. Mutations that inactivate this kinase w ere found in families with X-linked agammaglobulinaemia (XLA). In this stud y the Btk gene was analyzed in 13 registered Greek patients with XLA phenot ype originated from 12 unrelated families, in order to provide a definite d iagnosis of the XLA. The structure of Btk was analyzed at the cDNA level us ing the recently developed method, NIRCA (Non-Isotopic-Rnase-Cleavage-Assay ). Alterations were detected in all patients and sequencing analysis confir med the results and defined six novel XLA-associated Btk mutations (three m issense mutations: C337G, L346R, L452P; one nonsense mutation: Y392X, and t wo frameshift alterations: cl211-1212delA, c1306-1307insA). Having defined the genetic alteration in the affected males of these families, the informa tion was used to design polymerase chain reaction (PCR) primers and the Btk segments containing the mutated sequences were amplified from peripheral b lood derived genomic DNA of potential female carriers. The PCR products wer e directly sequenced and carrier status was determined in 12 out of 16 phen otypically normal females analyzed. This protocol can be used once the natu re of the Btk mutation has been defined in one of the affected males and pr ovides a convenient, simple and reliable way to determine the carrier statu s of other female family members. Molecular genetic analysis constitutes a determinative tool for the definitive diagnosis of XLA and may allow accura te carrier and prenatal diagnosis for genetic counselling.