Polymorphism and Utilization of Human VH Genesa

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Polymorphism and Utilization of Human V, Genesa

ERIC C. B. MILNER, WENDY 0. HUFNAGLE, ANNUSKA M. GLAS, IVY SUZUKI, AND

CATHERINE ALEXANDER Virginia Mason Research Center and

Department of Immunology University of Washington

Seattle, Washington

INTRODUCTION

The completion of the physical map of the V, I O C U S ’ ~ ~ has answered many questions and has raised a set of new ones. To a large extent, the issue now is to delineate allelic variation among V, genes and to determine the degree to which variation (or polymorphism) of V, genes contributes to differences in V gene utilization or to disease susceptibility or resistance. Polymorphic variation within the V, locus takes two fundamental forms. The first is conventional allelic variation in coding region sequences. Putative alleles of a number of genes have been identified by sequence analysis of the same gene isolated from different individuals. The second form of polymorphism and the more problematic to analyze is the result of numerous duplications, insertions, and deletions. This phenomenon leads to the unusual situation in which clearly distinct genes derived by duplication may be absolutely identical but may differ from their respective alleles by one or more nucleotide substitutions.

It is of considerable interest and importance to delineate the alleles of each V, gene as well as the haplotype structure including the insertion deletion polymorphisms. In the context of understanding the expressed antibody repertoire, it will be important to know and understand the germline repertoire. To address these issues, we have focused on the identification of specific genes and the discrimina- tion between closely related genes and alleles. We have used synthetic oligonucleo- tides corresponding to human V, sequences to analyze sequence variation in human genomic DNA. By employing conditions under which single nucleotide mismatches abrogate hybridization, it has been possible to unambiguously identify individual germline V, genes and to identify variation in closely related germline genes. We have demonstrated that analysis of the human V, locus with sequence- specific oligonucleotide probes reveals hybridization patterns of limited complexity and striking he te rogene i t~ .~?~ Within regions (FR1, CDRl, CDR2) different oligonucleotide probes are generally “mutually exclusive” by virtue of the nucleotide substitutions that distinguish the probes. Thus, a sequence detected by a given probe will not be detected by any mutually exclusive probe directed to the homolo-

a This research was supported in part by National Institutes of Health Grants AR39918,

Address for correspondence: Eric C. B. Milner, Virginia Mason Research Center, 1000 AI31241, and American Cancer Society Grant IM485.

Seneca St., Seattle, WA 98101. 50

MILNER et al.: POLYMORPHISM AND USE OF HUMAN VH GENES 51

gous region of a different V, gene segment. We have found that the detection of specific sequence motifs by hybridization of restriction enzyme digested DNA in a modified Southern analysis using short oligonucleotide probes allows the specific identification of germline gene^.^-^ Based on data obtained from genomic hybridization using such probes, it is possible to assign individualgenes to specific restriction fragments. By this approach, germline sequences of multiple individuals can be compared with a very high degree of precision.

POLYMORPHISM OF THE V d GENE FAMILY

A large number of germline sequences of VH4 genes have now been determined.6*s-*1 When the set of sequences is viewed as a whole, one striking feature of the heterogeneity that emerges is that most differences between any pair of sequences can be best described as the sharing or nonsharing of common conserved sequence motifs.6 The vH4 gene family could be resolved into 12 loci, 11 of which were located on chromosome 14 within the V, locus, and one located on chromosome 15.2f’J2,13 Hybridization with a full-length vH4 probe revealed 14 BglII restriction fragments12 (FIG. 1). Individual elements could be assigned to the BglII restriction fragments by sequence-specific oligonucleotide probe hybridiza- tied (FIG. 2). The various vH4 loci and the corresponding BglII bands are cata-

FIGURE 1. Detection of VH4 RFLP. DNA extracted from PBL derived from members of three families was digested with BglII, run on 1% aga- rose, and transferred to nylon mem- brane. Probing, washing, and exposure were performed as described in methods using the full-length VH4 probe. Bands are numbered from higher to lower MW. A poorly resolved triplet at band 9 is assigned both 9 and 10. Two rare fragments, designated IL and 6H, migrating just below and just above bands 1 and 6, are not present in the persons in this figure. Note that migration of DNA in lanes 1 and 4 is slightly retarded relative to the rest of the gel. Slow migration is characteristic of these DNA preparations and can be seen in lanes I and 4, FIGURE 3, as well. The upper panel is an 18-hr exposure; the lower panel is a three-day exposure included to enhance visibility of bands 13 and 14. Band 1, lane 4 is poorly visible in this figure but was easily visible on the original film. (Reprinted from Willerns van Dijk er a[.” with permission from The Journal of Immunology, 0 1991, The Journal of Immunology.)

52 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. Sequence-specific hybridization of VH4-specific oligonucleotide probes. Genomic DNA derived from donor 31 16 was digested with BglII and hybridized to the indicated oligonucleotide probes ( l o w s 1-13) or a cloned VH4 probe (lane 14). Compensation for differences in migration distance on the different gels used to generate this figure was accomplished by photographic enlargement or reduction to align the molecular weight markers. All hybridizations were performed at least twice, and comigration of elements was assessed by sequential hybridization of single gels. (Reprinted from van der Maarel et aL6 with permission from The Journal of Immunology, 0 1993, The Journal of Immunology.)

logued in TABLE 1. In the person studied, 9 of 11 loci were diploid, and two were single-copy insertion p o l y m o r p h i ~ m s . ~ ~ ~ ~ ~ ' ~ Alleles of the diploid locus, V4-61, could be discriminated by an RFLP as Bgl bands 8 and 11 .6 The remaining diploid loci comigrated. By sequence analysis and by sequence-specific hybridization, alleles of loci V4-28, carried on Bgl band 13, and V4-31, carried on Bgl band 10, could be discriminated. Bgl band 10 in this person carried two loci, V4-30.4 and V4-3 1. Bgt band 7 carried two chromosome 14 loci, V4-34 and V4-55, as well as the chromosome 15 locus. The alleles of V4-28 (band 13) differed by four nucleotides, resulting in two amino acid interchanges, a glutamine for an aspartic acid in FR1, and an isoleucine for a threonine in CDR2. The alleles of V4-31 differed by a single nucleotide in the first codon of framework 3, resulting in a leucine substitution for the conserved arginine at the beginning of framework 3. The apparent alleles of V4-61 (band 11 and band 8) differed profoundly. The band 8 allele differed from the band I 1 allele by 1 1 nucleotides resulting in five amino acid substitutions, a glutamine for a glutamic acid in FR1, an isoleucine for a valine in framework 1, alanine for a proline in FR2, and an arginine for a tyrosine and a threonine for a tyrosine in CDR2. It is possible that band 11 and band 8 are not true alleles, but rather represent insertion deletion polymorphisms that map to the same location.

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was found to carry the sequence of the VHsp germline gene. In rare individuals a duplication of the VHsP gene appears to have occurred, and, when present, the duplicated VHsp gene is carried on band 7 . The VHsp gene has not been formally assigned to one of the known loci but appears to map in the vicinity of V4-39.99'5 By sequence-specific hybridization, band 1 was not found to carry the sequence of VHsp in any person tested. By hybridization analysis the sequence carried in band 1 corresponds to the V4-4 sequence. The V4-4 sequence is one codon shorter in CDRl than V4-4b, and differs from V4-4b by 21 nucleotides. These nucleotide substitutions result in 12 amino acid interchanges.

No sequence has yet been assigned to band 3. In hybridization analysis band 3 does not hybridize to the commonly used oligonucleotide probes. The prevalence in the population of the BglII bands carrying vH4 genes is shown in TABLE 1 . Al- though the genes carried in band 6 and band 3 have not been mapped, they were found to be in significant linkage disequilibrium, suggesting that they are close together.'*

POLYMORPHISM OF VH3 GENES

At least 48 VH3 genes have been mapped to the VH Slightly more than half of these have been found to be pseudogenes.1,2 Variation among the vH3 genes is similar to that seen among the VH4 genes but is somewhat more difficult to delineate because of the larger number of elements. By assessing the occurrence of specific sequence motifs using oligonucleotide probes, however, it has been possible to identify many of the vH3 genes and to detect variation in their occurrence. We have analyzed the occurrence of several elements mapping in the vicinity of, or allelic to, V3-30 (designated l.9III,56pl1 h ~ 3 0 0 5 ) . ~ , ~ - ~ Hybridization analysis of these elements is shown in FIGURE 3. The results shown in FIGURE 3 in conjunction with a population analysis4 and deletion mapping studies5 indicate that all three elements cannot be alleles of the same locus. Calculations based on the prevalence of the individual elements 1.9III,56pl, and hv3005, and considering the known haplotype structures, indicate that 1.9111 (V3-30) and hv3005 (V3-30b) are appropriately considered to be alleles and 56pl (V3-30.3) an insertion/deletion element. Allele frequencies for hv3005 and 1.9111 are, respectively, 0.19 and 0.72. Because these allele frequencies do not add up to one, it seems likely that an additional haplotype or haplotypes with an allele frequency of 0.08 that lacks both 1.9111 and hv3005 occurs (i.e., a deletion of V3-30). No additional allelic sequence has been identified, however, suggesting that this putative allele is a blank haplotype. The putative third allele is definitely not 5 6 ~ 1 , which in our analysis always occurs on a haplotype carrying one or two copies of 1.9111.

We found that a SO-kb insertionideletion polymorphism profoundly affected the haplotype structure and allelic relationships in this r e g i ~ n . ~ The 50-kb insertion that carries 56pl and the second copy of I .9III has been localized to the interval between V3-30 and V4-31. There is some evidence for heterogeneity in the exact break points among the population. Using a probe adjacent to one of the S'I sites that border the insertion, we have found that approximately 93% of the population carry the insertion, an allele frequency of 0.73. At least six haplotypes can be identified in this region, four of which include the 50-kb insertion (FIG. 4). The occurrence of additional haplotypes is likely.

A second duplication and insertion/deletion involves the element V3-23 (also known as 3 0 ~ 1 ' ~ or VH2617). All persons thus far tested carry a copy of V3-23 on a 5-kb TuqI fragment. Approximately 40% of the population carries a duplicated

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FIGURE 4. Haplotype structures in the re ion between V4-28 and V3-31. Putative haplotypes were deduced from mapping s t u d i e ~ ' ~ ~ ~ ~ ~ ~ ' ~ and oligonucleotide probe a n a l y s i ~ . ~ . ~ . ~ . ~ ~

element on a 7-kb TuqI fragment. Phosphorimage analysis of band intensities indicate that the 5-kb band may carry alleles of both loci, only one of which is allelic to the 7-kb band. By deletional analysis in €3-cell lines, both of the V3-23 loci map to the same interval, suggesting that they may be tandemly arrayed.

Yet another V,3 duplication involves V3-53, (also known as 6 0 ~ 2 ' ~ ) . The V3-53 locus occurs on two sets of polymorphic TuqI fragments. The more 3' locus, which we have mapped by deletional analysis to the same interval as V3-53, is carried on TuqI fragments of approximately 3 kb and 4 kb. The second locus that maps 5' to the known V3-53 locus is carried on TaqI fragments of approximately 12 kb and 15 kb. No nucleotide or amino acid substitutions have as yet been associated with these RFLPs.

MILNER e l al.: POLYMORPHISM AND USE OF HUMAN VH GENES 57

VH1 POLYMORPHISM

The most complex allelic relationships that we have identified belong to the V1-69 locus. On the basis of sequence-specific hybridization, RFLP, or both, 13 variants that map to V1-69 are identified'* (FIG. 5). These variants encode seven distinct amino acid sequences. Only five codons are involved in the amino acid substitutions, one in CDRI, three in CDR2, and one in FR3 (FIG. 5). At each codon, a single amino acid interchange may be observed. These are a threonine for an alanine at position 33 in CDRI; an arginine for a glycine at position 50, a leucine for a phenylalanine at position 55, and an isoleucine for a threonine at position 57, all in CDR2; and a lysine for a glutamic acid at position 73 in FR3.18 Three haplotypes have been identified that carry duplications of the V1-69 gene. The most common duplication pairs variants 1 and 7 on a haplotype that occurs with a prevalence of 0.46 and a calculated allele frequency of 0.265 (FIG. 5). In the second duplication, variants 1 and 9 are paired on a haplotype that occurs with a prevalence of 0.04 and a calculated allele frequency of 0.0202. The third duplication pairs variants 2 and 6 on a haplotype that occurs with a prevalence of 0.02 and a calculated allele frequency of 0.0101. The sum of the allele frequencies for all variants, including duplications as single alleles, is 1.037, which is slightly greater than 1.0 and suggests that an additional duplication occurs that was unde- tected. The most likely duplication would be a haplotype pairing variants 5 and 11. Variant 11 occurred only in persons who carried variant 5, but in the population studied none of the three persons who carried variant 11 carried a third variant, as would be expected in the case of a duplication. However, these persons may have been homozygous for variant 5, because variant 5 occurs with a prevalence of 58% and variant 11 with a prevalence of just 6%. Taking into account this fourth putative duplication, the total allele frequencies equal 1.006, which is close to the expected value of 1.0.

HAPLOTYPE STRUCTURE

The existence of numerous insertionldeletion polymorphisms raises the possibility that different haplotypes of the V, locus may carry substantially different coding information. In addition, we and others have found little evidence for linkage disequilibrium between polymorphic elements in the VH locus. In several instances where linkage disequilibrium is evident, the elements involved have been physically closely linked, in several instances elements in linkage disequilibrium were carried on the same A-phage clone placing them within about 15 kb of one another. Inan analysis of 11 familiesin which42(of44) haplotypescould beidentified with respect to five loci, one of which is a VNTR located 5' to the J, region, 36 clearly distinct haplotypes could be identified. A group of three haplotypes were identical, and four haplotypes were ambiguous because they were uninformative at one or more loci (E. C. B. Milner and C. M. Alexander, unpublished observations). It seems likely that by analyzing additional markers, all 42 haplotypes would be found to be distinct. We have argued on the basis of similar observations that the haplotype structure in the population is extraordinarily ~ o m p l e x . ~ . ~ , ' ~

V H EXPRESSION AND POLYMORPHISM

Given the degree of polymorphism and the complexity of the VH locus, it becomes of considerable interest to know what influence these factors have on

58 ANNALS NEW YORK ACADEMY OF

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the composition of the antibody repertoire. It has been argued that the adult repertoire exhibits biased utilization of individual V, gene segments. Notably, the V3-23 gene segment (also called 3 0 ~ 1 , V,26, or 18/2) and the V4-34 gene segment (also designated 4.21) are overexpressed relative to other genes. 19-22 We have found that the repertoire in adult peripheral blood B cells exhibits intrinsic preferences for individual gene segments23 (FIG. 6). The repertoires of different persons exhibit preferences that presumably reflect their VH germline and genetic background, but which usually include V3-23 and V4-34 as predominant elements. These preferences could be the result of positive or negative selective processes occurring during B-cell differentiation, or could be the result of increased efficiency of rearrangement. In both V,3 and VH4 compartments, the genes represented most frequently among rearrangements, V3-23 and V4-34, are highly conserved elements that have not been found to be deleted in any subjects studied. The possibility that this reflects some evolutionary selection is uncertain. The nonfunc- tional gene, V4-55, has not been found to be deleted in any persons studied, and some elements that are highly expressed in persons possessing them have been found to be absent from the genome of other persons. An example of the latter is V4-3 1, and to a lesser extent V4-30.4, both of which are deleted in some persons, but which contribute significantly to rearrangements in persons possessing them.

One obvious insight made possible by the completion of the physical map of the V, locus is that the total number of functional V, genes is smaller than previously thought. Analyses of the expressed repertoire indicate that relatively few members of this, perhaps surprisingly, limited germline repertoire actually contribute to the expressed repertoire. In fact, we have found that fewer than eight VH3 genes make major contributions to the repertoire (each gene accounting for 8 to 20% of rearrangements), whereas the remainder of functional V,3 genes contribute at substantially lower levels (each gene accounting for 0 to 3% of

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B

L - 0 4 8 12 16 20

Percent of Total VH3 FIGURE 6. Utilization of VH3 repertoire in B cells from two subjects. The incidence of representation of eight specific VH3 gene segments in phagemid clones of quantitatively amplified rearranged VH3 genes from peripheral blood B cells (Subject 3 116, panel A ; Subject 4882, panel B ) . In panel A , 662 clones were analyzed, and in panel B , 142 clones were analyzed.


rearrangements) (FIG. 6 and W. 0. Hufnagle and E. C. B. Milner, unpublished observations). In a preliminary experiment in which both the germline copy number and the occurrence of rearrangement of individual VH genes in peripheral blood B cells were determined, it was found that the number of rearrangements of the preferentially expressed genes was roughly proportional to the copy number of those genes. Knowing the influence of the VH germline on the repertoire, it will now be of interest to determine the extent to which variation in the V, germline contributes to variation in immune responsiveness.

SUMMARY

The human VH germline repertoire comprises approximately 100 elements, which can be grouped into seven families based on nucleotide sequence similarity. Members of different families are interspersed throughout the complex, with limited sets of alleles identified for most loci. Linkage disequilibrium between most elements is weak. Variation within the population can be attributed to differences in nucleotide sequence between allelic genes as well as to differences in the number of genes present. Gene number per haplotype varies as a result of the common occurrence of insertion/deletion polymorphisms, which may be small, involving a single element, or may be extensive, involving four or five elements. In some cases, such polymorphisms may involve duplication of a functional V, gene segment on some haplotypes and deletion of the gene on others. The resulting variation in germline composition of the VH locus may have profound effects on V, gene utilization.

ACKNOWLEDGMENTS

We thank former members of the laboratory, especially Drs. KO Willems van Dijk and Eric H. Sasso, who have contributed immensely to these studies.

REFERENCES

1. MATSUDA, F., E. K. SHIN, H. NAGAOKA, R. MATSUMURA, M. HAINO, Y. FUKITA, S. TAKA-ISHI, T. IMAI, J. H. RILEY, R. ANAND, E. SOEDA & T. HONJO. 1993. Structure and physical map of 64 variable segments in the 3’ 0.8-megabase region of the human immunoglobulin heavy-chain locus. Nature Genet. 3: 88-94.

2. COOK, G. P., I. M. TOMLINSON, G. WALTER, H. RIETHMAN, N. P. CARTER, L. BULUWELA, G. WINTER & T. H. RABBITTS. 1994. Amap ofthe human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q. Nature Genet. 7: 162-168.

3. WILLEMS VAN DIJK, K., H. W. SCHROEDER, JR., R. M. PERLMUTTER & E. C. B. MILNER. 1989. Heterogeneity in the human immunoglobulin VH locus. J. Immunol.

4. SASSO, E. H., K. WILLEMS VAN DIJK & E. C. B. MILNER. 1990. Prevalence and polymorphism of human VH3 genes. J. Immunol. 145: 2751-2757.

5 . WILLEMS VAN DIJK, K., L. A. MILNER, E. H. SASSO & E. C. B. MILNER. 1992. Chromosomal organization of the heavy chain variable region gene segments compris- ing the human fetal antibody repertoire. Proc. Natl. Acad. Sci. USA 8 9 10430-10434.

6. VAN DER MAAREL, S. M., K. WILLEMS VAN DUK, C. M. ALEXANDER, E. H. SASSO,

142: 2547-2554.


7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

A. B. BULL & E. C. B. MILNER. 1993. Chromosomal organization of the human vH4 gene family: Location of individual gene segments. J. Immunol. 150: 2858-2868.

SASSO, E. H., K. WILLEMS VAN DWK, A BULL, S. M. VAN DER MAAREL & E. C. B. MILNER. 1992. VH genes in tandem array comprise a repeated germline motif. J. Immunol. 149: 1230-1236.

SANZ, I., P. KELLY, C. WILLIAMS, S. SCHOLL, P. TUCKER & J. D. CAPRA. 1989. The smaller human VH gene families display remarkably little polymorphism. EMBO J.

BAER, R., K.-C. CHEN, S. D. SMITH &T. H. RABBITTS. 1985 Fusionofan immunoglobulin variable gene and a T cell receptor constant gene in the chromosome 14 inversion associated with T cell tumors. Cell 43: 705-713.

WENG, N.-P., J. G. SNYDER, L.-Y. Yu-LEE & D. M. MARCUS. 1992. Polymorphism of human immunoglobulin vH4 germline genes. Eur. J. h n u n o l . 2 2 1075-1082.

VAN Es, J. H., M. HEUTINK, H. AANSTOOT & T. LOGTENBERG. 1992. Sequence analysis of members of the human Ig VH4 gene family derived from a single VH locus. Identification of novel germ-line members. J. Immunol. 149 492-497.

WILLEMS VAN DIJK, K., E. H. SASSO & E. C. B. MILNER. 1991. Polymorphism of the human immunoglobulin VH4 gene family. J. Immunol. 146: 3646-3651.

WILLEMS VAN DIJK, K., L. A. MILNER & E. C. B. MILNER. 1992. Mapping of human H chain V region genes (vH4) using deletional analysis and pulsed field gel electropho- resis. J. Immunol. 148 2923-2931.

WALTER, M. A., U. SURTI, M. H. HOFKER & D. W. Cox. 1990. The physical organization ofthe humanimmunoglobulin heavy chaingene complex. EMBO J. 9 3303-33 13.

BAER, R., A. FORSTER & T. H. RABBITTS. 1987. The mechanism of chromosome 14 inversion in a human T cell lymphoma. Cell 50: 97-105.

SCHROEDER, H. W., JR., J . L. HILLSON & R. M. PERLMUTTER. 1987. Early restriction of the human antibody repertoire. Science 238: 791-793.

MATTHYSSENS, G. & T. H. RABBITTS. 1980. Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region. Proc. Natl. Acad. Sci. USA

SASSO, E. H., K. WILLEMS VAN DIJK, A. BULL & E. C. B. MILNER. 1993. A fetally expressed VH1 gene belongs to a complex set of alleles. J. Clin. Invest. 91: 2358-2367.

HUANG, C. & B. D. STOLLAR. 1993. A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies. J . Immunol. 151: 5290-5300.

STEWART, A. K., C. HUANG, B. D. STOLLAR & R. S. SCHWARTZ. 1993. High-frequency representation of a single VH gene in the expressed human B cell repertoire. J. Exp. Med. 177: 409-418.

HUANG, C., A. K. STEWART, R. S. SCHWARTZ & B. D. STOLLAR. 1992. Immunoglobulin heavy chain gene expression in peripheral blood lymphocytes. J. Clin. Invest. 89:

PASCUAL, V., K. VICTOR, D. LELSZ, M. B. SPELLERBERG, T. J. HAMBLIN, K. M. THOMPSON, I. RANDEN, J. NATVIG, J. D. CAPRA & F. K. STEVENSON. 1991. Nucleo- tide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype. J . Immunol. 146: 4385-4391.

SUZUKI, I., L. PFISTER, A. GLAS, C. NOTTENBURG & E. C. B. MILNER. 1995. Represen- tation of rearranged VH gene segments in the human adult antibody repertoire. J . Immunol. In press.

8: 3741-3748.

77: 6561-6565.

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Polymorphism and Utilization of Human VH Genesa

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