Defect of BTK and block of B-cell differentiation. (A) Intracellular staining of BTK was performed and BTK expression was analyzed in peripheral blood CD14⁺ cells of the girl with agammaglobulinemia (left) and a healthy control (right). (B) The μ chain and VpreB were stained intracellularly. Analysis gate was set on CD19⁺ and/or VpreB-positive cells. In contrast to the normal B-cell development in the bone marrow of a healthy control (right), maturational block of B-cell precursors occurred and neither VpreB- μ + (low) nor VpreB- μ + (high) cells were observed in the patient (left). (C) The cDNA amplification was performed by PCR between exon 6 and exon 13 of BTK in the family members. Abnormally smaller-sized PCR products (1-3) were observed in the patient (lane 2) and her father (lane 5), although her paternal grandmother (lane 1), her mother (lane 3), and her brother (lane 4) expressed BTK mRNA with normal size. The primer pair was 5′-ATGCTATGGGCTGCCAAATT-3′ and 5′-GGTCCTTTGGATCAATTTCC-3′, and the expected size of the normal PCR product is 723 bp. M indicates marker. (D) The abnormally smaller-sized PCR products (1-3) in panel C were sequenced and found to be the result of splicing abnormality caused by the mutation of intron 11.