Normal somatic mutation frequencies and heavy-chain CDR3 repertoire in sorted IgD⁺CD27⁺ cells from IRAK-4– and MyD88-deficient patients. (A) Somatic mutations frequencies in the J_H4-J_H5 intron flanking rearranged V_HDJ_H4 sequences from sorted blood IgD⁺CD27⁺ and IgD⁻CD27⁺ cells of IRAK-4–, MyD88-, UNC-93B-, and TLR3-deficient patients (3.5 years) and young (< 4 years) or adult healthy controls (HC; see also supplemental Table 2). Mean values (expressed as mutations per 100 bp of total sequences) are indicated below the graph for each B-cell subset. IRAK-4– or MyD88-deficient patients and those deficient for UNC-93B or TLR3 were grouped. In control J_H4-J_H5 sequences, mutation frequencies gradually increased with age, reaching adult values for blood IgD⁺CD27⁺ B cells by approximately 4 years of age (S.W., J.-C. W and C.-A.R., unpublished data, 2009). (B) H-CDR3 spectratypes of the V_H3 transcripts expressed by sorted blood IgD⁺CD27⁺ and naive B cells of a healthy control and a MyD88- and an IRAK-4–deficient patient. V_H3-μ transcripts were prepared from 10 000 to 30 000 sorted cells, reverse transcribed, and amplified by PCR (see “Methods”). The PCR products were labeled by a run-off reaction with specific fluorescent V_H-FR3 consensus primers and subjected to capillary gel electrophoresis. As observed for naive cells, control IgD⁺CD27⁺ cells display a regular distribution of heavy-chain CDR3 sizes but showed a shift of 6 bp on average toward shorter CDR3 lengths. IgD⁺CD27⁺ cells from the IRAK-4– and the MyD88-deficient patient (P48 and P2) display a similar Gaussian distribution and average heavy-chain CDR3 size compared with control IgD⁺CD27⁺ cells (ie, a 6- or 7-bp mean difference in comparison with autologous naive cells). *The mean heavy-chain (H)–CDR3 length in amino acids (AA) inferred from the mean size of the PCR products is indicated. (H-CDR3 being defined as the region included between the invariant Cys and Trp residues of V_H and J_H genes, respectively.)