Representative Sanger sequencing traces for patients with BTK^Cys481variants. (A) Sanger sequencing traces on CD19-sorted BM cells showing BTK^{Cys481Arg(c.1634T>C)} and BTK^{Cys481Ser(c.1634T>A)} variants in patients WM2 and WM3, respectively. (B) Representative Sanger sequencing traces from cloning and sequencing studies show the presence of multiple BTK^Cys481 variants in patient WM2. Cloning and sequencing analysis showed 17/107 (15.9%), 21/107 (19.6%), and 7/107 (6.5%) clones expressed BTK^{Cys481Arg(c.1634T>C)}, BTK^{Cys481Ser(c.1634T>A)}, and BTK^{Cys481Ser(c.1635G>C)}, respectively, for patient WM2; whereas 2/119 (1.7%), 46/119 (38.7%), and 8/119 (6.7%) clones expressed BTK^{Cys481Arg(c.1634T>C)}, BTK^{Cys481Ser(c.1634T>A)}, and BTK^{Cys481Ser(c.1635G>C)}, respectively, for patient WM3. Arrows denote nucleotide variants. HD, healthy donor.