The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

Abstract

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.

FIGURE 1

Partial diplotypes of the IGHD locus, as determined for nine individuals. Only functional IGHD genes are shown. Genes that are highly similar (IGHD1-1, IGHD1-7, and IGHV1-20), and the very short IGHD7-27 gene are not shown, as these genes could not be distinguished with certainty in all individuals. Genes that are not rearrangeable (IGHD1-14, IGHD4-11, and IGHD6-25) are also omitted. IGHD5-5 and IGHD5-18 are identical sequences, and therefore one or both of the genes may be present where shown. Alleles are coded as shown in the key. Deletion polymorphisms are shown in black. Where a gene that was present in the genotype could not be identified in a haplotype with certainty, it is indicated as “?.”

FIGURE 2

Partial diplotypes of the IGHV locus, as determined for nine individuals. Only functional IGHV genes are shown. Rarely identified genes with published rearrangement frequencies of <0.5% (IGHV3-13, IGHV3-20, IGHV1-24*01, IGHV4-30-2, IGHV3-43, IGHV1-45, IGHV1-58, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, and IGHV1-f) are also omitted (22). IGHV4-59 and IGHV4-61 are also omitted as these genes could not be distinguished with certainty in all individuals. Alleles are color-coded as shown in the key. Where a gene that was present in the genotype could not be identified in a haplotype with certainty, it is indicated as “?.” Where a gene or allelic variant was known to be present in a genotype but insufficient sequences were available to allow confident haplotyping, no data are recorded. Ambiguities (e.g., *01 or *02) are indicated by vertically split cells, and duplications are shown by horizontally split cells. Apparent deletion polymorphisms are shown as gray cells and are marked “D.”

FIGURE 3

Mean rearrangement frequencies and SEMs of the IGHD3-9 and IGHD3-10 genes on chromosomes that carry the full set of 5′ IGHD genes (+) and on chromosomes where the genes from IGHD3-3 to IGHD2-8 appear to be deleted (−). Significant differences are indicated: **p < 0.01.