Repertoire diversity of V-DJ rearranged μ heavy chain transcripts. RT-PCR products from Figure 4 were separated on a denaturing 6% polyacrylamide (sequencing) gel. The control samples (lanes 1 and 2) were diluted 10-fold compared with the patient samples. PCR products from the μ heavy chain–deficient patient are shown in lane 3, and products from the Igα–deficient patient are shown in lane 4.

Flow cytometric analysis of B-cell development. Bone marrow mononuclear cells from a control (left column), μ heavy chain–deficient patient (middle column), and Igα-deficient patient (right column) were stained with antibody to CD19. Both patients showed a marked reduction in the number of CD19⁺ cells within the lymphoid gate (first row). Less than 1% of the CD19⁺ cells from either patient were B cells, as defined by coexpression of CD19 and surface immunoglobulin (second row). More than 75% of the B-lineage cells in both patients were positive for CD34 (third row). CD19⁺ permeabilized cells were stained for cytoplasmic TdT and μ heavy chain. More than 94% of the CD19⁺ cells from both patients were pro-B cells, as defined by coexpression of CD19 and TdT (fourth row).

Semiquantitative RT-PCR analysis of B cell–specific transcripts in the bone marrow. (a) Equal amounts of cDNA from 2 healthy controls (lanes 1 and 2), a patient with μ heavy chain deficiency (lane 3), the patient with Igα deficiency (lane 4), a cDNA negative template control (lane 5), and three 10-fold dilutions of control cDNA (10×, 1×, and 0.1×) (lanes 6–8) were amplified using primers specific for TdT, RAG1, VpreB, and λ5/14.1. GAPDH was used as a control to demonstrate equal concentrations of cDNA. (b) cDNA samples were analyzed as in a, except that the 10-fold dilutions in lanes 6–8 were 1×, 0.1×, and 0.01× of the control. Primers specific for all VH family members or primers specific for VH3, VH4, or VH1 were paired with a primer form the CH1 domain of μ heavy chain.

Identification of a mutation in the Igα gene. (a SSCP analysis of genomic DNA from a control (lane 1), patients with agammaglobulinemia (lanes 2–6), or no DNA template (lane 7) using primers specific for exon 3 of Igα. The DNA from the patient is shown in lane 3. (b) Sequence analysis revealed a single-bp substitution (A→G) at the invariant –2 position of the splice acceptor site for exon 3. The patient’s mutation is noted by an asterisk. (c) Schematic diagram of the 5 exons constituting the genomic structure of Igα. The extracellular region (open), the transmembrane region (TM; filled), the intracellular region (hatched), and the position of the point mutation in the patient are indicated.

Effects of the Igα mutation on splicing. (a) Bone marrow–derived cDNA from 2 healthy controls (lanes 1 and 2), a patient with μ heavy chain deficiency (lane 3), and the patient with mutations in Igα (lane 4) was amplified using primers from exon 2 and exon 5 of Igα (top) or primers specific for Igβ (bottom). Ten-fold dilutions of control cDNA (10×, 1×, and 0.1×) are shown in lanes 6–8. Sequencing of the smaller band in lane 4 demonstrated skipping of exon 3 of Igα. The sequence of the fainter band in this lane showed the use of a cryptic splice site within exon 3. (b) Schematic diagram showing exon skipping and activation of the cryptic splice site within exon 3. The coding sequence for exon 3 is shown within the central box. The majority of the Igα transcripts amplified in lane 4 in panel (a) delete exon 3, as indicated by the solid black line. A fraction of the transcripts are derived from the use of the cryptic splice site within exon 3, indicated by the dotted line. The invariant dinucleotides at the splice donor and acceptor sites are shown. The position of the point mutation and the dinucleotide at the cryptic acceptor site are underlined. The intron and the coding sequence that arise from the use of the cryptic splice site are shown in lowercase letters and uppercase letters, respectively.