Annotation Of Pseudogenous Gene Segments By Massively Parallel Sequencing Of Rearranged Lymphocyte Receptor Loci

The adaptive immune system generates a remarkable breadth of antigen-specific T cell receptors (TCRs) and B cell receptor (BCRs) by combinatoric shuffling of gene segments, enabling the immune system to recognize a diverse and unpredictable set of antigens. To generate this enormous diversity, lymphocytes undergo somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) region gene segments, which collectively encode the CDR3 region along with non-templated deletion or insertion of nucleotides at the V-D, and D-J junctions. Many of the V, D, and J gene segments at immune receptor loci are annotated as non-functional due to defects in primary sequence, motifs necessary for rearrangement, or chromosome position. However, full annotation of functional, pseudogene, or ORF, has proven elusive due to the random and unpredictable nature of non-templated deletions and insertions, the huge space of potential receptors, and the large V and J gene families.

We amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged immunoglobulin heavy chain (IGH) and T cell receptor beta (TCRB) receptor locus. Deep DNA sequencing allowed us to characterize the TCRB and IGH immune repertoire from mature T and B cells in a large cohort of healthy adults. Random chance predicts that less than one-third of somatic rearrangements at the TCRB and IGH loci will lead to transcripts with key motifs in-frame and no premature stops (i.e. a productive antigen receptor). Selection during lymphocyte maturation ensures that all mature T and B cells carry at least one rearrangement coding for a productive receptor, with a second allele rearranging in some cells that can be out of frame or include a V, D or J pseudogene segment (i.e. non-productive receptor). We classified each V, D, and J gene segment as functional or pseudogene based on the proportion of in-frame rearrangements and on 3-nt periodicity in the length of the CDR3 hypervariable coding region.

Based on these data we were able to conclusively annotate the functional status of each gene segment in the complex TCRB and IGH immune receptor loci. In TCRB, we found 2 presumed-functional gene segments that are clearly pseudogenes; in IgH we found 3 presumed-functional genes that are actually pseudogenes and 1 annotated pseudogene that is clearly functional. In both loci we identified numerous cases of gene segment functional/pseudogene status segregating among healthy subjects, indicating that genotype at the TCR and IGH loci may be a source of substantial differences in the naive antigen receptor repertoire between individuals. These results have helped us to more accurately characterize the genetic landscape underpinning somatic rearrangement of antigen receptors, a seminal event in the generation of robust antigen-specific immune responses.