DJ Pairing during VDJ Recombination Shows Positional Biases That Vary among Individuals with Differing IGHD Locus Immunogenotypes

Abstract

Human IgH diversity is influenced by biases in the pairing of IGHD and IGHJ genes, but these biases have not been described in detail. We used high-throughput sequencing of VDJ rearrangements to explore DJ pairing biases in 29 individuals. It was possible to infer three contrasting IGHD-IGHJ haplotypes in nine of these individuals, and two of these haplotypes include deletion polymorphisms involving multiple contiguous IGHD genes. Therefore, we were able to explore how the underlying genetic makeup of the H chain locus influences the formation of particular DJ pairs. Analysis of nonproductive rearrangements demonstrates that 3' IGHD genes tend to pair preferentially with 5' IGHJ genes, whereas 5' IGHD genes pair preferentially with 3' IGHJ genes; the relationship between IGHD gene pairing frequencies and IGHD gene position is a near linear one for each IGHJ gene. However, striking differences are seen in individuals who carry deletion polymorphisms in the D locus. The absence of different blocks of IGHD genes leads to increases in the utilization frequencies of just a handful of genes, and these genes have no clear positional relationships to the deleted genes. This suggests that pairing frequencies may be influenced by additional complex positional relationships that perhaps arise from chromatin structure. In contrast to IGHD gene usage, IGHJ gene usage is unaffected by the IGHD gene-deletion polymorphisms. Such an outcome would be expected if the recombinase complex associates with an IGHJ gene before associating with an IGHD gene partner.

FIGURE 1. Comparison of the frequency of (A) IGHD gene usage in productive and non-productive sequences (B) IGHJ gene usage in productive and non-productive sequences (C) IGHD gene usage in individuals with different IGHD genotypes (D) IGHJ gene usage in individuals with different IGHD genotypes

Het6 refers to IGHD gene usage in pooled rearrarangements from individuals who carried a single copy of a deletion polymorphism involving the six contiguous IGHD genes. Het5 refers to IGHD gene usage in pooled rearrarangements from individuals who carried a single copy of a deletion polymorphism involving the five contiguous IGHD genes. Rearrangements using each IGHD gene are shown as the proportion of the total number of rearrangements for which the IGHD gene could be identified. All genes are ordered as they appear in the genome, from 5′ (left) to 3′ (right). *p < 0.01. Error bars are SEM.

FIGURE 2. Proportion of rearrangements of each IGHD gene that pair with the four most frequently utilized IGHJ genes, using pooled data from the non-productive sequence datasets of 29 individuals

The proportion of rearrangements of each IGHD gene is plotted against chromosomal location, and the proportions are calculated with respect to the number of rearrangements in which the IGHD gene could be identified. As the genes are found in the genome in reverse orientation, the most 3′ gene (IGHD1-26) is shown at the extreme left and the most 5′ gene (IGHD2-2) is shown at the extreme right. ◆IGHJ3 IGHJ4 ▲IGHJ5 X IGHJ6

FIGURE 3. Consistency of rearrangement frequencies across four individuals who had no apparent IGHD gene deletions

Proportion shown are the proportions of all non-productive IGHJ4-containing (A) and IGHJ6-comtaining (B) rearrangements that include each IGHD gene; and the proportion of all non-productive IGHD3-10-containing (C) and IGHD3-22-containing (D) that include each of the four most highly utilized IGHJ genes (IGHJ3 – IGHJ6). Each individual is represented by a different symbol. As the IGHD and IGHJ genes are found in the genome in reverse orientation, the most 3′ gene (IGHD1-26) is shown at the extreme left and the most 5′ gene (IGHD2-2) is shown at the extreme right. The most 3′ IGHJ gene (IGHJ6) is shown at the extreme left and the most 5′ gene (IGHJ3) is shown at the extreme right.

FIGURE 4. IGHD gene utilization frequencies in individuals with different IGHD genotypes

Proportions of rearrangements using each IGHD gene were compared between four individuals who carried complete sets of IGHD genes on both their chromosomes, with (A) pooled non-productive sequence data for two individuals who carried a single copy of a deletion polymorphism involving the six contiguous IGHD genes, IGHD3-3 to IGHD2-8 (Het6); and (B) pooled non-productive sequence data for three individuals who carried a single copy of a deletion polymorphism involving the five contiguous IGHD genes, IGHD3-22 to IGHD1-26 (Het5). Proportions of IGHD genes are a proportion of the total number of rearrangements in which the IGHD gene could be identified. IGHD genes are ordered as they appear in the genome, from 5′ (left) to 3′ (right). *p < 0.00001. **p < 1.0×10⁻⁹.