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Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences.

Duvvuri B, Wu GE - Front Immunol (2012)

Bottom Line: We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients.GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids.The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

View Article: PubMed Central - PubMed

Affiliation: School of Kinesiology and Health Science, Faculty of Health, York University Toronto, ON, Canada.

ABSTRACT
Gene conversion (GCV), a mechanism mediated by activation-induced cytidine deaminase (AID) is well established as a mechanism of immunoglobulin diversification in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and the presence of all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23(*)01 gene sequences from common variable immunodeficiency (CVID) patients, AID-deficient patients, and healthy individuals to survey "GCV-like" activities. We analyzed rearranged IGHV3-23(*)01 gene sequences obtained from total PBMC RNA and single-cell polymerase chain reaction of individual B cell lysates. Our search identified strong evidence of GCV-like activity. We observed that GCV-like tracts are flanked by AID hotspot motifs. Structural modeling of IGHV3-23(*)01 gene sequence revealed that hypermutable bases flanking GCV-like tracts are in the single stranded DNA (ssDNA) of stable stem-loop structures (SLSs). ssDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV3-23-(*)01 sequences from healthy individuals compared to that of CVID patients. We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients. GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

No MeSH data available.


Related in: MedlinePlus

Gene conversion-like events between rearranged IGHV3-23*01 gene sequences and germline VH sequences superimposed by somatic hypermutation (SHM). IGHV3-23*01 mutated sequences obtained by single-cell PCR from CD19+IgM+B cells (Brezinschek et al., 1995, 1997; Dörner et al., 1998; Longo et al., 2008). Topmost sequence is the germline of rearranged IGHV3-23*01 gene sequences from healthy individuals (X87050.1, X87049.1, and X87051.1) Potential donor germline IGHV3 family gene segments are shown below the rearranged IGHV3-23*01 sequences. GCV-like tract is underlined. Flanking SHM/AID hotspot motifs are underlined and italicized.
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Figure 2: Gene conversion-like events between rearranged IGHV3-23*01 gene sequences and germline VH sequences superimposed by somatic hypermutation (SHM). IGHV3-23*01 mutated sequences obtained by single-cell PCR from CD19+IgM+B cells (Brezinschek et al., 1995, 1997; Dörner et al., 1998; Longo et al., 2008). Topmost sequence is the germline of rearranged IGHV3-23*01 gene sequences from healthy individuals (X87050.1, X87049.1, and X87051.1) Potential donor germline IGHV3 family gene segments are shown below the rearranged IGHV3-23*01 sequences. GCV-like tract is underlined. Flanking SHM/AID hotspot motifs are underlined and italicized.

Mentions: To identify GCV-like events in rearranged IGHV3-23*01 gene sequences, we searched the germline human IGVH locus data bases for matches to the mutated IGHV3-23*01 gene sequences. The IGHV3-23*01 gene sequences were collected from data bases of sequences from healthy individuals, CVID, and AID-deficient patients, Figure 1 shows statistically significant regions of GCV-like events identified by GENECONV in rearranged IGHV3-23*01 gene sequences, that had undergone affinity maturation as evidenced by the presence of SHM (henceforth referred to as hypermutated IGHV3-23*01 gene sequences). Figure 1 contains the analysis of IGHV3-23*01 gene sequences extracted from a healthy individual and a CVID patient, total PBMC RNA; Figures 2–4 contains analysis of IGHV3-23*01 gene sequences extracted from single-cell PCR of B cells from healthy individuals (details in Materials and Methods). No evidence of GCV-like events was identified in from IGHV3-23*01 sequences extracted from single-cell PCR of B cells from AID-deficient patients. Germline VH donors, tract length, and P-values from GENECONV are shown in Tables 2 and 3. The observed base substitutions in GCV-like events show clustered nucleotide differences, an unvarying characteristic of GCV, and thus striking evidence for GCV. Inspection of the IGVH locus2 revealed that both upstream and downstream germline VH genes were involved in the diversity of rearranged IGHV3-23*01 gene sequences through GCV-like mechanisms. Germline VH gene donors downstream of IGHV3-23*01 are: IGHV3-9*01, IGHV3-11*01, IGHV3-19*01, and IGHV3-21*01. Germline VH gene donors upstream of IGHV3-23*01 are: IGHV3-43*02, IGHV3-48*03, IGHV3-60*01 (a pseudogene), and IGHV3-64*04. All germline donor VH genes are functional unless so noted. The implication of these results is discussed below.


Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences.

Duvvuri B, Wu GE - Front Immunol (2012)

Gene conversion-like events between rearranged IGHV3-23*01 gene sequences and germline VH sequences superimposed by somatic hypermutation (SHM). IGHV3-23*01 mutated sequences obtained by single-cell PCR from CD19+IgM+B cells (Brezinschek et al., 1995, 1997; Dörner et al., 1998; Longo et al., 2008). Topmost sequence is the germline of rearranged IGHV3-23*01 gene sequences from healthy individuals (X87050.1, X87049.1, and X87051.1) Potential donor germline IGHV3 family gene segments are shown below the rearranged IGHV3-23*01 sequences. GCV-like tract is underlined. Flanking SHM/AID hotspot motifs are underlined and italicized.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375636&req=5

Figure 2: Gene conversion-like events between rearranged IGHV3-23*01 gene sequences and germline VH sequences superimposed by somatic hypermutation (SHM). IGHV3-23*01 mutated sequences obtained by single-cell PCR from CD19+IgM+B cells (Brezinschek et al., 1995, 1997; Dörner et al., 1998; Longo et al., 2008). Topmost sequence is the germline of rearranged IGHV3-23*01 gene sequences from healthy individuals (X87050.1, X87049.1, and X87051.1) Potential donor germline IGHV3 family gene segments are shown below the rearranged IGHV3-23*01 sequences. GCV-like tract is underlined. Flanking SHM/AID hotspot motifs are underlined and italicized.
Mentions: To identify GCV-like events in rearranged IGHV3-23*01 gene sequences, we searched the germline human IGVH locus data bases for matches to the mutated IGHV3-23*01 gene sequences. The IGHV3-23*01 gene sequences were collected from data bases of sequences from healthy individuals, CVID, and AID-deficient patients, Figure 1 shows statistically significant regions of GCV-like events identified by GENECONV in rearranged IGHV3-23*01 gene sequences, that had undergone affinity maturation as evidenced by the presence of SHM (henceforth referred to as hypermutated IGHV3-23*01 gene sequences). Figure 1 contains the analysis of IGHV3-23*01 gene sequences extracted from a healthy individual and a CVID patient, total PBMC RNA; Figures 2–4 contains analysis of IGHV3-23*01 gene sequences extracted from single-cell PCR of B cells from healthy individuals (details in Materials and Methods). No evidence of GCV-like events was identified in from IGHV3-23*01 sequences extracted from single-cell PCR of B cells from AID-deficient patients. Germline VH donors, tract length, and P-values from GENECONV are shown in Tables 2 and 3. The observed base substitutions in GCV-like events show clustered nucleotide differences, an unvarying characteristic of GCV, and thus striking evidence for GCV. Inspection of the IGVH locus2 revealed that both upstream and downstream germline VH genes were involved in the diversity of rearranged IGHV3-23*01 gene sequences through GCV-like mechanisms. Germline VH gene donors downstream of IGHV3-23*01 are: IGHV3-9*01, IGHV3-11*01, IGHV3-19*01, and IGHV3-21*01. Germline VH gene donors upstream of IGHV3-23*01 are: IGHV3-43*02, IGHV3-48*03, IGHV3-60*01 (a pseudogene), and IGHV3-64*04. All germline donor VH genes are functional unless so noted. The implication of these results is discussed below.

Bottom Line: We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients.GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids.The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

View Article: PubMed Central - PubMed

Affiliation: School of Kinesiology and Health Science, Faculty of Health, York University Toronto, ON, Canada.

ABSTRACT
Gene conversion (GCV), a mechanism mediated by activation-induced cytidine deaminase (AID) is well established as a mechanism of immunoglobulin diversification in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and the presence of all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23(*)01 gene sequences from common variable immunodeficiency (CVID) patients, AID-deficient patients, and healthy individuals to survey "GCV-like" activities. We analyzed rearranged IGHV3-23(*)01 gene sequences obtained from total PBMC RNA and single-cell polymerase chain reaction of individual B cell lysates. Our search identified strong evidence of GCV-like activity. We observed that GCV-like tracts are flanked by AID hotspot motifs. Structural modeling of IGHV3-23(*)01 gene sequence revealed that hypermutable bases flanking GCV-like tracts are in the single stranded DNA (ssDNA) of stable stem-loop structures (SLSs). ssDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV3-23-(*)01 sequences from healthy individuals compared to that of CVID patients. We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients. GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

No MeSH data available.


Related in: MedlinePlus