The Human Immunoglobulin VH Gene Repertoire Is Genetically Controlled and Unaltered by Chronic Autoimmune Stimulation
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The Human Immunoglobulin VH Gene Repertoire is Genetically Controlled and Unaltered by Chronic Autoimmune Stimulation Hitoshi Kohsaka,* Dennis A. Carson,§ Laura Z. Rassenti,§ William E.R. Ollier,ʈ Pojen P. Chen,§ Thomas J. Kipps,§ and Nobuyuki Miyasaka‡ *Division of Immunological Diseases, Medical Research Institute, and ‡First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, 113, Japan; §Department of Medicine, Sam and Rose Stein Institute for Research on Aging, University of California, San Diego, La Jolla, California 92093; and ʈARC Epidemiology Research Unit, University of Manchester, Manchester, M13 9PT, United Kingdom Abstract consists of 22 functional genes. The VH1 and VH4 families each contain approximately a dozen functional genes, and VH2, The factors controlling immunoglobulin (Ig) gene repertoire VH5, VH6, and VH7 families contain 3, 2, 1, and 1 functional formation are poorly understood. Studies on monozygotic genes, respectively (1). twins have helped discern the contributions of genetic ver- Although human fetal B lymphocytes rearrange and ex- sus environmental factors on expressed traits. In the present press a highly restricted set of VH genes (2–6), the frequency of experiments, we applied a novel anchored PCR-ELISA sys- Ig VH gene rearrangement in cord blood lymphocytes is tem to compare the heavy chain V gene (VH) subgroup rep- roughly proportional to VH family size (7). In adults, some ertoires of and ␥ expressing B lymphocytes from ten pairs genes such as VH 18/2, and VH 4.21 are expressed in high fre- of adult monozygotic twins, including eight pairs who are quency (8–10). Deletions or duplications of individual VH concordant or discordant for rheumatoid arthritis. The re- genes may partially explain their relative expression through a sults disclosed that the relative expression of each Ig VH gene dosage effect (11). However, inherited differences in reg- gene subgroup is not precisely proportional to its relative ulatory or coding sequences and/or environmental stimuli genomic size. The monozygotic twins had more similar IgM could also influence VH gene expression. VH gene repertoires than did unrelated subjects. Moreover, Previous studies on T cell receptor variable region gene monozygotic twins who are discordant for RA also use (TCRV) alleles revealed that apparently minor sequence poly- highly similar IgM VH gene-subgroup repertoires. Finally, morphisms can affect gene expression in the periphery (12– the VH gene repertoire remained stable over time. Collec- 14). By analyzing monozygotic adult twins, we disclosed that tively, these data reveal that genetic factors predominantly the TCRV gene repertoires of ␣ T lymphocytes are con- control VH gene repertoire formation. (J. Clin. Invest. 1996. trolled mainly by genetic factors. In contrast, the repertoire of 98:2794–2800.) Key words: monozygotic twins • B cell • ␥␦ lymphocytes depends principally on environmental stimuli rheumatoid arthritis • PCR • ELISA (15). HLA is the predominant genetic factor in TCRBV reper- toire formation, whereas polymorphisms of TCRV genes Introduction probably play a lesser role (16–18). The goals of the present investigations were to ascertain B cells produce immunoglobulins (Ig) as effector molecules the genetic regulation and stability of human Ig VH gene ex- and as antigen receptors. The mature Ig molecule is the prod- pression, and to determine if a chronic immunologic disease, uct of multiple non-contiguous gene segments. The variable rheumatoid arthritis (RA), could unbalance the repertoire. regions of Ig H chains are assembled from variable (VH), di- Both and ␥ transcripts were analyzed from 10 pairs of versity (DH) and joining (JH) genes. The human VH complex is monozygotic twins, including two normal pairs, four pairs dis- composed of approximately 100 gene segments per haploid ge- cordant, and four pairs concordant for RA. The problem of Ig nome, including at least 51 functional genes, as judged by suc- VH gene diversity was avoided by application of a quantitative cessful rearrangement in cloned cDNA. The remainder are ei- anchored PCR-ELISA technology (19, 20). The results showed ther pseudogenes or potentially functional genes never found that the expression of VH genes in adults was not proportional productively rearranged. On the basis of nucleic acid sequence to VH gene family size. The VH3 and VH4 gene families were homology, the VH genes have been grouped into 6–7 families. dominant in and ␥ transcripts, while the large VH1 gene fam- Among the seven families, the VH3 family is the largest, and ily was underrepresented. Regardless of the presence or ab- sence of RA, the peripheral B cells from monozygotic twins had homologous and distinctive patterns of VH family gene ex- Address correspondence to Hitoshi Kohsaka, M.D., Division of Im- pression. Also, the V gene-subgroup expression pattern for munological Diseases, Medical Research Institute, Tokyo Medical H each individual remained stable, over 6 month period of obser- and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113, Ja- pan. Phone: 81-3-5803-5819; FAX: 81-3-5684-0717 or 81-3-5684-0057; vation. Thus, genetic factors primarily control VH family gene E-mail: [email protected] repertoire formation in humans. The inherited pattern of VH Received for publication 28 May 1996 and accepted in revised form expression is not grossly altered by time, or chronic systemic 15 October 1996. autoimmune disease. J. Clin. Invest. Methods © The American Society for Clinical Investigation, Inc. 0021-9738/96/12/2794/07 $2.00 Clinical samples. Monozygotic twins with RA were identified in the Volume 98, Number 12, December 1996, 2794–2800 course of epidemiological investigations on RA in the United King- 2794 Kohsaka et al. Table I. RA Monozygotic Twins Studied tion (0.5–3 kb) and hydrolysis of the remaining RNA, they were poly-G tailed with dGTP and terminal deoxytransferase. Subsequently, free Duration dGTP was removed by two sequential sodium acetate precipitation Twin Age Sex of disease HLA DR steps. The dG-tailed first strand cDNA were subjected to primary APCR amplification in 100 l of 10 mM Tris-HCl (pH 8.3), 50 mM Discordant twins KCl, 1.5 mM MgCl2, 0.001% gelatin, 200 M dNTPs, 1 U Perfect A1 65 F 25 yr 4/10 Match Polymerase Enhancer (Stratagene, La Jolla, CA), 2.5 U Taq A2 65 F No disease 4/10 DNA polymerase (Boehringer Mannheim, Indianapolis, IN). The B1 44 F 9 yr 3/7 primers consisted of anchor primers (9:1 mixture of AN and ANC B2 44 F No disease 3/7 primers) and C1 for IgM heavy chain V domain cDNA amplifica- C1 47 M No disease 2 tion, or C␥1 for IgG heavy chain V domain cDNA amplification, at a C2 47 M 1 yr 2 concentration of 0.5 M. The amplification consisted of 20 cycles of D1 64 F No disease 2/11 95ЊC for 30 s, 42ЊC for 30 s, and 72ЊC for 1 min followed by final ex- tension for 7 min. The products were size selected (500–750 bp) on D2 64 F 14 yr 2/11 agarose gels and one third was removed for nested PCR. The reaction Concordant twins mixture was same as that of the primary anchored PCR except for the E1 55 F 19 yr 1/6 primers; 0.5 M each of an AN primer and a BTNC2 or BTNC␥2 E2 55 F 11 yr 1/6 primer were used. The biotinylated primers specified upstream re- gions of the C1 or C␥1 priming sequence. Twenty-five cycles of F1 39 F 19 yr 4 nested PCR were carried out at an annealing temperature of 53ЊC, F2 39 F 19 yr 4 and the products were purified on mini-columns (Wizard PCR preps; G1 57 F 27 yr 1/6 Promega, Madison, WI). Nested PCR reactions with the biotinylated G2 57 F 22 yr 1/6 primer were performed not only to increase the specificity of the am- H1 42 F 19 yr 1/4 plification but also to put a biotin molecule at the 5Ј end of the anti- H2 42 F 24 yr 1/4 sense strand of the PCR product. Construction of VH1-6 leader DNA. To standardize hybridization efficiencies of the six probes, a 124-bp DNA fragment which con- All affected twins were seropositive. Homozygosity for HLA-DRB tained tandemly aligned leader specific sequences of each of six Ig V shared epitope was a potent risk factor for disease concordance (38). H gene subgroups was generated. A pool of the six VH-family specific oligonucleotides and bridging oligonucleotides (VH1/2: 5Ј- ACA AAG TAT CCT CCA GGT, VH2/3: 5Ј- CAA ACT CAG GAG CCT dom (21). Two normal pairs were identified in Tokyo and San Diego. GGA, VH3/4: 5Ј- GCC ACC AGC CAG CTC AGC C, VH4/5: 5Ј- GCG They were all Caucasians except for one pair of Japanese twins. AGG ATT CTG GGA GCT, VH5/6: 5Ј- AGG AGA CAC AGG Monozygosity of the studied twins was confirmed by genotype analy- AGG AGG. VH6/H; 5Ј- ACC AAG CTT GGA AGA TGA GGA sis using anonymous probes. RA was diagnosed according to the [underlined is a HindIII restriction site]) were mixed, phosphorylated American College of Rheumatology criteria (22). The affected twins and ligated. The ligated DNA fragment was amplified with an E/VH1 were seropositive for rheumatoid factors. The clinical profiles of the primer (5Ј- CCA GTC CAT GAA TTC AGA [underlined is a EcoRI RA monozygotic twins are shown in Table I. The patients had active restriction site]) and a VH6/H primer using Pfu DNA polymerase disease when their blood samples were collected. Peripheral blood (Stratagene), and subcloned into pBluescript II SKϩ plasmid (Strat- mononuclear cells (PBMC) were isolated with density gradient sedi- agene).