Computational Inference, Validation, and Analysis of 5’UTR-Leader Sequences of Alleles of Immunoglobulin Heavy Chain Variable Genes

Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5’-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5’-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions.

Computational inference, validation, and analysis of 5'UTR-leader sequences of alleles of immunoglobulin heavy chain variable genes

Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5'-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5'-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions.

Immunoglobulin T genes in Neopterygii

In teleost fishes there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD) and T (IgT). IgT has been the last to be described and is considered a teleosts-fish specific isotype. From the recent availability of genome sequences of fishes, an in-depth analysis of Actinopterygii immunoglobulin heavy chain genes was undertaken. With the aid of a bioinformatics pipeline, a machine learning software, CHfinder, was developed that identifies the coding exons of the CH domains of fish immunoglobulins. Using this pipeline, a high number of such sequences were obtained from teleosts and holostean fishes. IgT was found in teleost and holostean fishes that had not been previously described. A phylogenetic analysis reveals that IgT CH1 exons are similar to the IgM CH1. This analysis also demonstrates that the other three domains (CH2, CH3 and CH4) were not generated by recent duplication processes of IgM in Actinopterygii, indicating it is an immunoglobulin with an earlier origin.