Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V kappa gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease.

Navajos and genetically related populations have a 10-fold increased incidence of Haemophilus influenzae type b (Hib) disease compared with control populations. The Vkappa gene A2 is used to encode the majority of anti-Hib Abs, and these are the highest affinity anti-Hib Abs. Navajos carry a different allele of the A2 gene segment (A2b) that is defective in its ability to undergo V-J recombination. The A2b allele has only three nucleotide changes from the commonly occurring A2a allele, two of which could potentially affect its ability to recombine. In this study we used two independent in vitro assays to test whether the nucleotide change found in the A2b promoter and/or in the A2b recombination signal sequence (RSS) might be responsible for the decrease in recombination frequency observed in vivo. Using a luciferase reporter gene assay, we found no significant difference between A2a and A2b promoter activities. However, the competition recombination substrate assay showed a 4.5-fold reduction in the relative frequency of recombination of the A2b RSS compared with A2a. We show that this decreased frequency is due to a synergistic effect of the unique nucleotide change present in the heptamer of the A2b RSS and the shared nucleotide change present in the nonamer of both A2b and A2a. This in vitro relative frequency of rearrangement is not significantly different from that observed in vivo; therefore, the A2b RSS is probably the factor associated with the increased susceptibility to Hib disease among individuals carrying the A2b allele.

Sequence of the spacer in the recombination signal sequence affects V(D)J rearrangement frequency and correlates with nonrandom Vkappa usage in vivo.

Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vkappa gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.

Human cord blood kappa repertoire.

To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease.

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.