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Polymorphisms in human immunoglobulin heavy chain variable genes and their upstream regions.

ABSTRACT: Germline variations in immunoglobulin genes influence the repertoire of B cell receptors and antibodies, and such polymorphisms may impact disease susceptibility. However, the knowledge of the genomic variation of the immunoglobulin loci is scarce. Here, we report 25 potential novel germline IGHV alleles as inferred from rearranged naïve B cell cDNA repertoires of 98 individuals. Thirteen novel alleles were selected for validation, out of which ten were successfully confirmed by targeted amplification and Sanger sequencing of non-B cell DNA. Moreover, we detected a high degree of variability upstream of the V-REGION in the 5'UTR, L-PART1 and L-PART2 sequences, and found that identical V-REGION alleles can differ in upstream sequences. Thus, we have identified a large genetic variation not only in the V-REGION but also in the upstream sequences of IGHV genes. Our findings provide a new perspective for annotating immunoglobulin repertoire sequencing data.

SUBMITTER: Mikocziova I

PROVIDER: S-EPMC7261178 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Polymorphisms in human immunoglobulin heavy chain variable genes and their upstream regions.

Mikocziova Ivana I Gidoni Moriah M Lindeman Ida I Peres Ayelet A Snir Omri O Yaari Gur G Sollid Ludvig M LM

Nucleic acids research 20200601 10

Germline variations in immunoglobulin genes influence the repertoire of B cell receptors and antibodies, and such polymorphisms may impact disease susceptibility. However, the knowledge of the genomic variation of the immunoglobulin loci is scarce. Here, we report 25 potential novel germline IGHV alleles as inferred from rearranged naïve B cell cDNA repertoires of 98 individuals. Thirteen novel alleles were selected for validation, out of which ten were successfully confirmed by targeted amplifi ...[more]

PMID: 32365177

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Project description:Extensive diversity has been identified in the human heavy chain immunoglobulin locus, including allelic variation, gene duplication, and insertion/deletion events. Several genes have been suggested to be deleted in many haplotypes. Such findings have commonly been based on inference of the germline repertoire from data sets covering antibody heavy chain encoding transcripts. The inference process operates under conditions that may limit identification of genes transcribed at low levels. The presence of rare transcripts that would indicate the existence of poorly expressed alleles in haplotypes that otherwise appear to have deleted these genes has been assessed in the present study. Alleles IGHV1-2*05, IGHV1-3*02, IGHV4-4*01, and IGHV7-4-1*01 were all identified as being expressed from multiple haplotypes, but only at low levels, haplotypes that by inference often appeared not to express these genes at all. These genes are thus not as commonly deleted as previously thought. An assessment of the 5' untranslated region (up to and including the TATA-box), the signal peptide-encoding part of the gene, and the 3'-heptamer suggests that the alleles have no or minimal sequence difference in these regions in comparison to highly expressed alleles. This suggest that they may be able to participate in immunoglobulin gene rearrangement, transcription and translation. However, all four poorly expressed alleles harbor unusual sequence variants within their coding region that may compromise the functionality of the encoded products, thereby limiting their incorporation into the immunoglobulin repertoire. Transcripts based on IGHV7-4-1*01 that had undergone somatic hypermutation and class switch had mutated the codon that encoded the unusual residue in framework region 3 (cysteine 92; located far from the antigen binding site). This finding further supports the poor compatibility of this unusual residue in a fully functional protein product. Indications of a linkage disequilibrium were identified as IGHV1-2*05 and IGHV4-4*01 co-localized to the same haplotypes. Furthermore, transcripts of two of the poorly expressed alleles (IGHV1-3*02 and IGHV4-4*01) mostly do not encode in-frame, functional products, suggesting that these alleles might be essentially non-functional. It is proposed that the functionality status of immunoglobulin genes should also include assessment of their ability to encode functional protein products.

Project description:Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Dataset Information

Polymorphisms in human immunoglobulin heavy chain variable genes and their upstream regions.

Publications

Polymorphisms in human immunoglobulin heavy chain variable genes and their upstream regions.

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