Non-functional immunoglobulin G transcripts in a case of hyper-immunoglobulin M syndrome similar to type 4

Abstract

Summary 86% of immunoglobulin G (IgG) heavy-chain gene transcripts were found to be non-functional in the peripheral blood B cells of a patient initially diagnosed with common variable immunodeficiency, who later developed raised IgM, whereas no non-functionally rearranged transcripts were found in the cells of seven healthy control subjects. All the patient's IgM heavy-chain and kappa light-chain transcripts were functional, suggesting that either non-functional rearrangements were being selectively class-switched to IgG, or that receptor editing was rendering genes non-functional after class-switching. The functional gamma-chain sequences showed a normal rate of somatic hypermutation while non-functional sequences contained few somatic mutations, suggesting that most came from cells that had no functional gene and therefore were not receiving signals for hypermutation. However, apoptosis of peripheral blood lymphocytes was not impaired. No defects have been found in any of the genes currently known to be responsible for hyper-IgM syndrome but the phenotype fits best to type 4.

Figure 1

FACS plots of gated lymphocytes of the patient and a healthy control showing expression of IgM and IgG on CD19-positive cells (B cells).

Figure 2

Structure of the patient's VDJ junctions in IgG heavy-chain clones indicating the best-matched V_H, D and J_H segments to parts of the numbered sequences. The codon numbering is the official numbering, not the actual number of codons from the beginning of V_H. (Codon 95 is the 99th codon; see text). Codons of the J_H are only numbered if in frame. = V_H, = D, = J_H (stripes indicate correct frame, gaps indicate actual frame reading through from V_H– thus the above example is out of frame), and unhighlighted letters = junctional bases.

Figure 2

Structure of the patient's VDJ junctions in IgG heavy-chain clones indicating the best-matched V_H, D and J_H segments to parts of the numbered sequences. The codon numbering is the official numbering, not the actual number of codons from the beginning of V_H. (Codon 95 is the 99th codon; see text). Codons of the J_H are only numbered if in frame. = V_H, = D, = J_H (stripes indicate correct frame, gaps indicate actual frame reading through from V_H– thus the above example is out of frame), and unhighlighted letters = junctional bases.

Figure 3

HCDR3 sequences of the patient's IgM transcripts. *For these sequences no D match was found. ‡D match tentative only; not counted as D in Table 3. Mutated bases are shown in bold type.

Figure 4

Categories of base substitutions in SHM in V regions of all immunoglobulin transcripts from the patient. Substitutions that were present in two or more clonally related sequences were only counted once. The figures in the grid are actual numbers, not percentages.

Figure 5

Investigation of apoptosis and cell death of PBMC of patient and control by propidium iodide staining to measure DNA content. (a) Examples of raw data from FACS analysis, showing numbers of cells against PI fluorescence, and percentages of cells falling into the diploid (live) region 1, apoptotic, 2, and dead, 3. For each plot 50 000 cells were counted. These plots are for all cells present. Region 1 was defined by gating viable lymphocytes. The rest of the range to the left of that, covering cells in the process of DNA degradation, is somewhat arbitrarily divided between apoptotic cells and ones that have little DNA left and are dead. These plots (in 5a) are for cells cultured 5 days in complete medium with no added stimulant. (b) Histograms of the percentages of cells apoptotic and dead at day 0 and after 5 and 8 days culture with either no stimulant or pokeweed mitogen (PWM) or dexamethasone. For details see Materials and methods. N.B. These results are percentages not total numbers. The higher percentages of apoptotic and dead cells in the control than in the patient at Day 8 reflect the fact that, amongst the patient's cells, most cells that were committed to apoptosis had completely disappeared by this stage, leaving higher percentages of surviving cells viable.