Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment

Abstract

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.

Fig. 1.

Position of primers on a partial representation of the Homo sapiens IGHG genes. The CH1 exon is not shown. H represents the single hinge exon of IGHG1, IGHG2 and IGHG4, and the most 3′ hinge exon of IGHG3 H4 (Gene tables > IGHC, IMGT Repertoire, http://www.imgt.org). A focus on the intron separating CH2 and CH3-CHS exons is also shown. The primers are represented by arrows which indicate their position and 5′ > 3′ sequence orientation. Sequence numbering positions are for the allele*01 of each IGHG gene (IMGT/LIGM-DB Accession numbers (24): J00228 for IGHG1, J00230 for IGHG2, X03604 for IGHG3, and K01316 for IGHG4) according to IMGT/BlastSearch (http://www.imgt.org). Black arrows represent primers used for PCR and sequencing: N°1/IGHG1_1017–1036 (FWD 5′-gccgggtgctgacacgtcca-3′), N°2/IGHG1_1820–1801 (REV 5′-cttgccggccgtcgcactca-3′), N°3/IGHG2_1018–1037 (FWD 5′-gctgggtgctgacacgtcca-3′), N°4/IGHG2_1819-1800 (REV 5′-cttgcyggccgtggcactca-3′), N°5/IGHG3_1596–1615 (FWD 5′-gtcgggtgctgacacatctg-3′), N°6/IGHG3_2400–2381 (REV 5′-cttgccggcyrtsgcactca-3′), N°7/IGHG4_1029–1048 (FWD 5′-gcatccacctccatctcttc-3′), N°8/IGHG4_1820–1801 (REV 5′-cttgccggccctggcactca-3′). Dark gray arrows represented IGHG consensual sequencing primers, they are common for the 4 IGHG genes: N°9/IGHGseqF (FWD 5′-aggtcagcctgacctgcctg-3′) localized 1544–1563 for IGHG1, 1543–1562 for IGHG2, 2124–2143 for IGHG3, and 1544–1563 for IGHG4, and N°10/IGHGseqR (REV 5′-tggagaccttgcacttgtac-3′) localized 1336–1317 for IGHG1, 1334–1315 for IGHG2, 1915–1896 for IGHG3, and 1335–1316 for IGHG4. Light gray arrows represented IGHG specific sequencing primers: N°11/IGHG1_1390–1405 (FWD 5′-acccgtggggtgcgag-3′), N°12/IGHG1_1405–1390 (REV 5′-ctcgcaccccacgggt-3′), N°13/IGHG2_1058–1073 (FWD 5′-ccacctgtggcaggac-3′), N°14/IGHG2_1388–1403 (FWD 5′-acccgcggggtatgag-3′), N°15/IGHG3_1999–2014 (FWD 5′-aggccagcttgaccca-3′), N°16/IGHG3_2010–1995 (REV 5′-tcaagctggcctctgt-3′), N°17/IGHG4_1348–1366 (FWD 5′-cgtcctccatcgagaaaac-3′), N°18/IGHG4_1356–1340 (REV 5′-tggaggacgggaggcct-3′). FWD = forward; REV = reverse; r = a or g; y = c or t; s = g or c.