X-chromosome inactivation and mutation pattern in the Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinemia

Background: The diagnosis of X-linked agammaglobulinemia (XLA) is not alway s clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in th e gene for Brutton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA pheno type. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic coun seling. Materials and Methods: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the fram ework of the Italian Multicenter Clinical Study on XLA. We used non-isotopi c RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. Results: Using the cDNA-based approach, the identification of BTK gene abno rmalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infa nts. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identifed. In s poradic cases, BTK gene abnormalities were identified in 9 out of 10 patien ts whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrand om pat tern of XCI had mutations of the BTK gene. Conclusions: Our findings indicate that in sporadic cases BTK gene sequenci ng is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multip le generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.