The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pan, Q.
Right arrow Articles by Hammarström, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pan, Q.
Right arrow Articles by Hammarström, L.
The Journal of Immunology, 1998, 161: 3520-3526.
Copyright © 1998 by The American Association of Immunologists

Characterization of Human {gamma}4 Switch Region Polymorphisms Suggests a Meiotic Recombinational Hot Spot Within the Ig Locus: Influence of S Region Length on IgG4 Production1

Qiang Pan, Hodjattallah Rabbani and Lennart Hammarström2

Division of Clinical Immunology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human {gamma}4 gene RFLPs, revealed after BamHI digestion, show IGHG4 alleles of 9.0 (9.2), 9.4, and 9.6 kb at various frequencies in different ethnic populations. Studies in immunodeficient individuals have previously suggested that the 9.4 BamHI allele is associated with a higher serum level of IgG4 than the 9.0 (9.2) BamHI allele, but it is not clear whether this is associated with the S region itself or other control elements. In addition, a duplication of the 9.4-kb {gamma}4 allele has recently been observed in a high proportion of normal donors. We therefore undertook a study of the structural basis for the difference in Ab levels in the various {gamma}4 alleles. We demonstrate that the S{gamma}4 alleles differ in length due to deletions and insertions of a varying number of 79-bp S{gamma}4 repeat units. Two novel RFLPs, 8.8 and 9.1 kb, were also observed. The alleles are likely to be generated by unequal crossing over, and the breakpoints cluster in S{gamma}4 repeat units that contain chi-like motifs, implicating chi-like sequences in the meiotic recombination. Our data support the idea that the 9.4-kb BamHI allele is more productive than the 9.0 (9.2)-kb allele in normal healthy donors, possibly due to the extended switch regions, whereas duplication of the {gamma}4 gene has no effect on switching and IgG4 serum levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human Ig heavy chain constant gene locus (IGHC)3 is composed of nine functional genes and two pseudogenes, located on the long arm of chromosome 14 (1). The genes are organized into two blocks, IGHG3-IGHG1-IGHEP1-IGHA1 and IGHG2-IGHG4-IGHE-IGHA2 (2, 3), and numerous types of deletions and duplications of single or multiple constant region genes have been observed in different populations (4). Considerable genetic variation in this locus has been observed both at the protein level (Gm and A2 m allotypes) and at DNA level (RFLP for the G, E, and A genes; the Sµ, S{gamma}, and S{alpha} regions; and the I (intervening) {gamma} and I{alpha} regions). Many of these polymorphisms are suggested to be due to unequal crossing over during meiosis, but the sequence motifs or structures involved in this process are as yet unknown. Additional mechanisms for generation of polymorphism include short insertions/deletions or the presence or the absence of a given restriction site as a consequence of a point mutation (5).

The {gamma}-gene RFLPs produced by BamHI can be used as markers for the IGHGP, IGHG2, and IGHG4 genes. IGHG4 alleles of 9.0, 9.2, 9.4, and 9.6 kb have all been described at various frequencies in different ethnic populations (1, 4, 5). These polymorphisms have provided valuable tools for determining the organization of the IGHC locus and for a variety of population genetics and immunologic investigations.

Human IgG4 Abs are functionally monovalent (6) and do not, under normal circumstances, activate complement. Although IgG4 only represents a minor portion of total IgG, it may nevertheless be of clinical importance, as IgG4-deficient individuals have been reported to suffer from recurrent infections (7). Furthermore, elevated levels of IgG4 Abs are often noted against selected protein Ags after chronic exposure (8), and this Ab subclass is involved in a variety of allergic diseases (9, 10).

Isotype switching is the process by which a B cell alters the heavy chain class or subclass of the Abs produced. The switch recombination process occurs between tandemly repeated sequences in the switch (S) regions located upstream of the heavy chain constant region genes. The length of the S region has been hypothesized to influence serum levels of the IgG subclasses in the mouse (11, 12, 13), whereas a completely different mechanism was recently described for human IgG3, where the differential switching between the b and g allotypes appears to be due to point mutations in a crucial NF-{kappa}B binding motif in one of the S{gamma}3 repeats (14).

In humans, the 9.4 BamHI allele has been suggested to be more productive than the 9.0 BamHI allele based on data obtained from IgG4-deficient individuals (15) and IgA-deficient individuals carrying the H1 haplotype (1) of {gamma} genes (16), respectively. The molecular basis for the {gamma}4 gene RFLPs has previously not been identified. It is, therefore, unclear whether the effect noted is associated with the S region itself or with other control elements upstream of the S{gamma}4 region (15). In addition, the 9.4 BamHI allele is preferentially associated with a 370-kb rather than a 350-kb MluI fragment that was recently found to carry a tandem duplication of the {gamma}4 gene (17). As both {gamma}4 genes in the duplicated haplotype are transcriptionally active, this may confound previous conclusions based on serum IgG4 levels. We therefore undertook a study of the structural basis for the differential regulation of Ab synthesis in the various RFLP alleles of human IgG4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA preparation

Genomic DNA from individuals from different ethnic populations (described in 4 was prepared using previously described methods (4, 14). The genomic DNA samples were digested by different restriction enzymes and electrophoresed in a 1% agarose gel; the areas containing S{gamma}4, based on expected sizes, were excised; and DNA was purified using a Gel Quick DNA purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

RFLP and Southern blot analysis

PFGE, RFLP, and Southern blot analysis for detection of C{gamma} regions was performed as described (4). The probe for S{gamma} was a 1.2-kb SalI-NcoI fragment, derived from a pGEM-T clone containing a S{gamma}2 region amplified by PCR from a healthy blood donor. The probe for C{gamma} was a 2.0-kb HindIII-EcoRI fragment from clone 24B (4).

PCR amplification

S{gamma}4 region amplifications. The primers and the conditions for PCR amplification of the S{gamma} region have been described previously (14). Briefly, 500 ng of genomic DNA or 3 µl of 20 µl of purified DNA (recovered from the restriction enzyme-digested gel slice described above) dissolved in a 25-µl volume was amplified for a total of 30 cycles consisting of 1 min at 94°C, 1 min at 69°C, and 3 min at 72°C. Taq DNA polymerase (Promega, Madison, WI) was used in all experiments, and the error rate of this enzyme was reported previously as 3 in 10,000 (18). Cosmids CosIg6, CosIg8, and CosIg13, which contain the different human {gamma} genes (3), were used as positive and negative controls.

Sµ-S{gamma}4 fragment amplifications. To amplify the Sµ-S{gamma}4 fragments directly from genomic DNA, we designed a {gamma}4-specific primer based on our S{gamma}4 sequence data (EMBL database, accession no. Y12547, Y12549, and Y12552) and used it in a nested PCR in which we initially amplified all µ-{gamma} switch fragments using the Sµ1 and S{gamma}common primers, followed by a second round using another µ primer (Sµ5) together with the {gamma}4-specific primer, S{gamma}4C (5'-tcgtcgacGATGCTCTCCCCTGTTTC; Fig. 1Go). A salI restriction site (underlined) was introduced in this primer. In the first run, we used 500 ng of DNA in a 10-µl reaction and amplified the mixture for a total of 35 cycles consisting of 50 s at 94°C, 50 s at 63°C, and 2 min at 72°C. Products (0.5 µl) from the first run were used in the second run (25-µl reaction) and amplified for 40 cycles (50 s at 94°C, 50 s at 68°C, and 1 min at 72°C)



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 1. Diagram of the primers used in the nested PCR. We initially amplified all µ-{gamma} switch fragments with primers Sµ1 and S{gamma}c followed by a second round using the µ primer (Sµ5) together with the S{gamma}4-specific primer (S{gamma}4c). The arrows indicate the approximate positions of the primers.

 
Sequencing and sequence analysis

PCR products were purified, ligated into the pGEM-T vector, and transformed into JM109 competent cells according to the manufacturer’s instructions (Promega). TaqFS dye terminator cycle sequencing was performed using an automated fluorescent sequencer (AB1, 373A-Stretch, Perkin-Elmer) from Cybergene (Huddinge, Sweden). The primers used for sequencing were T7, Sp6, Hs{gamma}4as1 (5'-CCAGCTCCTGCCAAAATCTA-3'), and Hs{gamma}4as4 (5'-TCTGATGCTCTCCCCTGTTTC-3'). Sequence data analysis was performed using the LASERGENE software package (DNASTAR, Madison, WI).

Determination of IgG subclass levels

Serum samples were obtained from the individuals described previously (4), and levels of IgG subclasses were determined by radial immunodiffusion according to the manufacturer’s instructions (The Binding Site, Birmingham, U.K.). The specificity of the anti-subclass reagents has been demonstrated previously (19, 20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Different numbers of S region repeat units cause the polymorphism in the {gamma}4 locus

To locate the differences that determine the RFLP alleles of the human {gamma}4 locus, we used BamHI-, HindIII-, and PstI-digested genomic DNA of individuals from different populations in Southern blot analyses using a S{gamma} probe. As shown in Table IGo, the differences between the BamHI {gamma}4 RFLP alleles are consistent with the results of HindIII, BamHI-HindIII, and BamHI-PstI digestions, which all indicate that the differences in size are located within the fragment containing the S{gamma}4 region (Fig. 2Go). In addition to the recognized BamHI RFLP alleles of 9.0 (designated 9.2 by us), 9.4, and 9.6 kb, two new alleles, an 8.8-kb allele in the Mongoloid population and a 9.1-kb allele in both the Caucasoid and Mongolian populations were observed (Table IGo and Fig. 2Go, B andC).


View this table:
[in this window]
[in a new window]
  		Table I. RFLP allele size of the human S{gamma}4 regions

 


View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. Southern blot analysis of human S{gamma}4 regions. A, Strategy of Southern blot analysis and PCR amplification of the human S{gamma}4 region. To be able to detect whether the difference between the S{gamma}4 regions caused the difference between the BamHI alleles, a S{gamma} probe was used in the Southern blot analysis. The 3' BamHI site is located about 9 kb downstream of the 5' BamHI site, and the RFLP alleles that result from this enzyme digestion will include the S{gamma}4 and C{gamma}4 regions. The HindIII-digested fragment contains the I{gamma}4 and S{gamma}4 regions, whereas the fragment derived from double digestion by BamHI and PstI only contains the S{gamma}4 region. The positions of the primers (P1 and P2) that were used to amplify the S{gamma}4 region in a separate set of experiments are indicated by arrows. B and C, An S{gamma} probe was used to visualize the S region-containing bands, and only the positions of S{gamma}4 alleles are indicated. Lane 1 shows the result from a Swedish individual who has been shown to carry the 9.4-kb BamHI allele. Lanes 2 and 4, A Swedish and a Gambian individual who carry the 9.2-kb BamHI alleles. Lane 3, A Japanese individual with 9.6-kb BamHI alleles. Lane 5, A Chinese individual with 9.2- and 9.1-kb alleles. B, HindIII digestion. C, BamHI-PstI double digestion of the same samples.

 
To determine whether the differences in size were due to alterations in the S region itself, we used PCR to amplify the S{gamma}4 regions directly from genomic DNA. Since the primers used amplify all four S{gamma} regions (14), and the expected sizes of S{gamma}2 and S{gamma}4 are similar (1.2 and 1.1 kb, respectively) (14, 21), we could not directly identify all the different bands visualized in these experiments.

Based on the previous Ig locus map, the BamHI site upstream of the S{gamma}2 region is located more than 10 kb further upstream than the corresponding site 5' of the S{gamma}4 region (3), whereas the 3' HindIII sites are localized in approximately the same position (Fig. 2Go). We therefore double-digested genomic DNA with BamHI-HindIII and subjected it to gel electrophoresis, and the area between 1.8 and 2.9 kb was cut out and purified. The DNA samples from the excised part of the gel were subjected to the PCR described above, and the products were expected to include all the S{gamma}4 variants (Table IGo), but not the S{gamma}2-containing segments. We then cloned all the PCR products, and partial sequencing showed that 11 of 13 fragments contained the S{gamma}4 region from different alleles, whereas the remaining two were S{gamma}2, indicating that there may exist a polymorphic BamHI site immediately 5' of the S{gamma}2 region.

We subsequently determined the complete sequences of the S{gamma}4 region in 11 individuals with differing lengths of S region. Although there is a high overall similarity between the nine 79 mer repeats that constitute the S{gamma}4 region, as previously described by Mills et al. (21), there exists a sufficient number of nucleotide substitutions to allow identification of a given repeat. Our S{gamma}4 sequences differ from the published sequence (21) in two respects. First, there is a 79-bp insertion within the described sixth repeat (21) in all our sequences except the 8.8-kb allele (which lacks a number of repeats), thus changing the alignment and numbering of the 3' repeat units. In addition, there are nine positions where all our 11 sequences share the same nucleotide but that differ from the published sequence (21). Based on the alignment of the 79 mer repeats defined by our sequences and as shown in Figure 3Go, S{gamma}4 regions of the different BamHI {gamma}4 loci differ in length due to deletions and insertions of a varying number of 79-bp S{gamma}4 repeats.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 3. Aligned human S{gamma}4 region repeat units from individuals carrying the different BamHI alleles. Each sequence was derived from one allele of a single individual. The EMBL accession numbers are Y12549 (Japanese 9.6 kb), Y12547 (Swedish 9.4 kb), Y12548 (Gambian 9.2 kb), Y12551 (Chinese 9.1 kb), and Y12550 (Japanese 8.8 kb). The chi-like motifs suggested to be associated with recombination in human cells (22, 23) are underlined.

 
The prototype S{gamma}4 region sequence in our experiments contains ten 79 mer repeat units, was found in both the Negroid (Gambian) and Caucasian (Swedish) populations, and would be expected to correspond to the 9.0-kb BamHI RFLP allele described previously (1). As the sizes of these alleles have not as yet been determined accurately, we propose to call this allele 9.2. The 9.1 and 9.4 alleles show a loss of one repeat unit and an addition of two repeat units, respectively (Fig. 4GoA). The additional alleles observed (9.6- and 8.8-kb BamHI) in the Mongolian population (Chinese and Japanese) may have arisen due to unequal crossing over, resulting in a gain of four repeat units and a deletion of five repeat units, respectively (Fig. 4GoB). The breakpoint in the 9.4-kb allele mapped to nucleotide 10 in the 5 repeat. The breakpoints in the other S{gamma}4 alleles mapped to the 2, 3, 4, 5, 7, and 8 repeats. Although they all center around the 10th nucleotide in the repeat, they could not be mapped with certainty due to the high sequence similarity in this region. However, they occur within a region 2 to 20 bp upstream or 15 bp downstream of the number 10 nucleotide of the repeat, suggesting a hot spot within the S{gamma}4 region for meiotic recombination. Interestingly, the 3, 4, and 8 repeat units contain a chi-like motif (CC(T/A)CC(T/A)GC; i.e., GC(A/T)GG(A/T)GG on the opposite strand) previously found in the recombination hot spot region of the bcl-2 proto-oncogene (22) and human I{alpha}1 region (23); the 4, 5, and 8 repeat units contain another chi-like motif with a 1-bp substitution; and 2 and 7 repeat units contain a number of chi-like motifs, although less well conserved (75%).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. Schematic structure of human S{gamma}4 region. S{gamma}4 regions of individuals who carry the different BamHI {gamma}4 locus differ in lengths due to deletions and insertions of a varying number of 79-bp S{gamma}4 repeat units. The boxes filled with different colors represent the different 79-bp repeat units. Arrows denote four to six nucleotide changes compared with the prototype S{gamma}4 region (9.2 kb), whereas changes of three or fewer nucleotides are not marked. The number at the right end indicates the number of samples that have been sequenced. A, Comparison of the S{gamma}4 regions from 9.4-, 9.2-, and 9.1-kb alleles. B, Comparison of the S{gamma}4 regions from 9.6-, 9.2-, and 8.8-kb alleles.

 
The frequencies of the 9.0 (9.2), 9.4, and 9.6 alleles in different populations were summarized previously (4). The S{gamma}4 region of the new 8.8 allele gives rise to a 760-bp PCR product, as ascertained by sequencing (Fig. 3Go), and has a frequency in the Mongoloid population of 29.2% (n = 130). It is almost exclusively (35 of 38 samples) restricted to donors carrying a duplication of the IGHA1-IGHE genes, which is present in approximately 40% of the Mongoloid population (4). However, not all individuals with this type of duplication carry the 760-bp S{gamma}4 region, as only 35 of 57 tested samples were positive. As this {gamma}4 allele could be confused with or masked by the Caucasian 8.8-kb BamHI {psi}{gamma} allele, additional probes, such as S{gamma}, should be used when assessing RFLP patterns of human {gamma} genes.

No difference in frequencies of different S{gamma}4 RFLP alleles were found between the Chinese and Japanese populations (data not shown). No 760-bp S{gamma}4 fragment was seen in the 50 Caucasian DNA samples tested, suggesting that this allele is restricted to the Mongoloid population.

Influence of the length of the {gamma}4 switch region on serum levels of IgG4

The 9.4-kb BamHI allele has previously been suggested to be more productive than the 9.0 (9.2)-kb BamHI allele in immunodeficient individuals, i.e., resulting in a higher serum level of IgG4 in the former (15, 16). This may argue in favor of an influence of the length of the switch region on serum levels of IgG4. This superiority could be confirmed in Caucasian (Swedish) and Mongoloid (Chinese and Japanese) healthy blood donors with a normal number of {gamma}4 genes, i.e., carrying the 350-kb MluI allele (Table IIGo). Individuals carrying the 9.6-kb BamHI allele did not exhibit higher levels of serum IgG4 than donors with the 9.0 (9.2)- or 9.4-kb alleles (Table IIGo). However, the wide range of IgG4 serum levels and the limited number of donors available (n = 6) preclude a definite conclusion on the role of the switch region of the 9.6-kb allele.


View this table:
[in this window]
[in a new window]
  		Table II. IgG4 serum levels in donors homozygous for different BamHI RFLP alleles1

 
Influence of the number of {gamma}4 genes on serum level of IgG4 and switch frequency

Using PFGE, two MluI RFLP alleles of 350 and 370 kb, respectively, can be distinguished in the Caucasian population (4, 24) and represent approximately 60 and 40%, respectively, of the donors tested (4). Although the 350-kb band can be associated with all {gamma}4 RFLP alleles, the 370-kb MluI band is exclusively associated with the 9.4-kb BamHI RFLP allele. Recently, it was suggested that the 370-kb MluI fragment contains a tandem duplication of the IGHC4 gene (17). This was confirmed by densitometric measurements of {gamma} genes in Southern blot in our own donors (Fig. 5Go).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 5. Duplication of the human IGHC4 gene in individuals carrying the 370-kb MluI fragment. A, PFGE analysis from four normal Caucasian donors. DNA was digested with MluI and hybridized with the IGHC4 probe. Lanes 1 and 2 are from individuals who are 350 kb homozygous, and lanes 3 and 4 are from 350 to 370 kb heterozygous individuals. B, Southern blot analysis of the same individuals. DNA was digested with BamHI and hybridized with the IGHC4 probe. Compared with lane 1, showing an individual heterozygous for the 9.4- and 9.2-kb (previously denoted 9.0) IGHC4 fragments, lanes 3 and 4 show an extra copy of the 9.4-kb IGHC4 fragment.

 
As duplication of a block of IGCH genes, including the {gamma}4 gene, may result in an elevated level of serum IgG4 (19), we determined the serum levels of IgG4 in Caucasian donors (as the 370-kb MluI band is absent in the Mongolian population) homozygous for the 9.4-kb BamHI allele and either of the two MluI alleles. As can be seen in Table IIIGo, there was no significant difference between the groups, suggesting a limited or no effect of the number of {gamma}4 genes on IgG4 serum levels.


View this table:
[in this window]
[in a new window]
  		Table III. Influence of duplication of the {gamma}4 gene on IgG4 serum levels in Caucasian donors1

 
The serum concentration of IgG is regulated by a feedback mechanism involving the FcRn (25), and although we did not observe an influence of the duplicated {gamma}4 gene on total IgG4 levels, an effect on switching itself may thus not be readily demonstrable. Genomic DNA samples from PBMC from five pairs of donors were therefore subjected to nested primer PCR to determine switch frequencies. Each pair of samples was run at the same time under identical conditions. The number of Sµ-S{gamma}4 fragments was enumerated from 10 PCR reactions, with each DNA sample run in parallel. Sixteen of these Sµ-S{gamma}4 fragments were sequenced (data not shown; the accession numbers for the breakpoints sequences are AF057497–AF057518, GenBank). Although there was an overall correlation between serum IgG4 levels and the number of switch fragments, there was no difference when samples from individuals with similar serum IgG4 levels but differing number of {gamma}4 genes were compared (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The {gamma}4 gene and its switch region exhibit extensive polymorphism (1, 5, 15, 26, 27), which is now proven to be due to deletions and duplications within the switch region. The mechanism is likely to be unequal crossing over, although other mechanisms, such as looping out excision and gene conversion, cannot be ruled out. The breakpoints cluster in S{gamma}4 repeat units that contain one or two chi-like motifs. This motif was previously described as a recombinational hot spot within the major breakpoint region of the bcl-2 locus (22, 28) and the human intervening {alpha}1 region (23), implicating this motif in translocations and recombinations within the Ig locus.

The chi sequence is well known for directing recombination in prokaryotic cells through the action of the RecBCD enzyme (29). The chi sequence has also been shown to be involved in the generation of repetitive sequences in phylogenetically distantly related organisms (30) and is present at a high frequency within the Ig genes of the mouse (31). However, whether this sequence or related sequences affect recombination in eukaryotic cells has not been proven.

Another interesting candidate for a role in generation of the different S{gamma}4 alleles is Rad51, a highly conserved eukaryotic homologue of the prokaryotic recombination protein RecA. The mouse and human Rad51 homologues are 83% identical with the yeast protein, and mammalian Rad51 has been strongly correlated with meiotic recombination, DNA repair, and switch recombination (32, 33, 34). It has previously been shown that the yeast Rad51 preferentially binds GT-rich DNA that shares significant homology with genetically unstable eukaryotic genomic sequences, including human microsatellites and Alu repetitive elements (35). The repetitive G-rich S region may thus be one of the additional targets for a class of recombinational enzymes that includes Rad51.

An increase in the serum level of IgG4 in individuals with duplicated {gamma}4 genes has been previously reported (19, 36). However, a large proportion of the samples tested were derived from individuals with duplications of multiple Ig constant region genes and would thus be expected to contain additional copies of the recently described enhancer located between the {alpha}1 and {psi}{gamma} genes (37, 38, 39). In both the human and mouse Ig loci, the region 3' of the C{alpha} gene(s) contains four tissue-specific DNase I-hypersensitive sites. It was previously shown that murine plasmacytoma clones stably transfected with an HS1234-linked c-myc construct, expresses c-myc in a copy number-dependent manner, suggesting that HS1234 may function as a locus control region (40). This region may also serve to maintain the IgH constant genes in a transcriptionally competent chromatin structure as well as to regulate the isotype switching of the constant region genes through interactions with their specific promoter in normal B cells. Deletion of this enhancer is associated with a low serum level of IgG4 (20), and it is thus possible that the increase in IgG4 levels that we described previously (19), could be due to the effect of increased enhancer activity. However, as we previously did not find any increase in the levels of IgG2 in individuals with a heterozygous {gamma}2 duplication, some of which would be expected to encompass the enhancer region (19, 36), it would have to be postulated that this effect would be limited to the IgG4 subclass. It may therefore be of interest to study the interactions between the different enhancer elements and the different {gamma} gene promoters.

Considerable effort was used to determine the in vivo switch frequency to the {gamma}4 gene, but due to the limitation of the PCR method (where the small fragments were preferably amplified), the number of the switch fragments generated by our nested PCR strategy can at best be considered an approximate in vivo switch frequency. However, we did not find an influence of the number of the {gamma}4 genes on switch frequency. As the switch frequency correlated roughly to the IgG4 serum level, it is still possible that since human Ig gene expression is highly regulated in vivo, the effect of multiple genes on switching may be regulated by other factors that may affect the 3' enhancer and/or the I{gamma}4 promoter region.

It is currently not clear how the length of the S{gamma}4 region (or other genetic control elements within the respective alleles) influences the switching process and, subsequently, IgG4 production. One possibility is that differences in the number and the fine structure of the S{gamma}4 repeat units could affect the switching by altering the efficiency with which individual repeat units are used as substrates for the recombination machinery and by altering the number of recombination targets. Moreover, the whole structure of a given S{gamma}4 may affect transcription. A direct strategy to confirm the influence of switch region length could be to use a DNA substrate that can undergo switch recombination in vitro (41, 42, 43, 44, 45, 46, 47).

In addition to the S{gamma}4 polymorphisms described in this paper, previously published data (27, 48) suggest the existence of 3.7-, 3.9-, and 4.1-kb HindIII alleles, providing support for the presence of additional S{gamma}4 regions with different numbers of repeat units. The S{gamma}4 sequence of Mills and co-workers contains nine repeat units (21), but the missing part differs from that in our 9.1-kb BamHI allele, thus adding to the complexity of this region.

Despite the constant length and organization of the S{gamma}3 regions associated with both the b and g allotypes (14), marked differences are noted in the rate of switching (49) and in the resulting serum IgG3 levels (50). These differences appear to be due to point mutations in a crucial NF-{kappa}B binding motif in one of the S{gamma}3 repeats (14). IgG4 deficiency has been shown to be associated with both the S{gamma}4 and, stronger still, a region upstream of the S{gamma}4 (15) that contains the I{gamma}4 and I{gamma}4 promoter. As transcription of the I{gamma}4 region is a necessary step preceding the switch itself, it is possible that in addition to the differences in length and structure of S{gamma}4, structural alterations in the I{gamma}4 and I{gamma}4 promoter regions may influence the frequency of switching and thus the serum levels of IgG4. As there are no major differences in the I{gamma}3 and I{gamma}3 promoter regions in individuals with normal and low levels of IgG3 (14, 51), this suggests that despite the rather recent duplication of the human {gamma} genes, the switch process may be differently regulated among the human IgG subclasses.


   Acknowledgments
 
We are indebted to Prof. J. Stavnezer for carefully reviewing the manuscript. We thank Prof. Gerald R. Smith for discussions on the involvement of chi sequences in recombinational processes.


   Footnotes
 
1 This work was supported by the Swedish Medical Research Council. Back

2 Address correspondence and reprint requests to Dr. Lennart Hammarström, Department of Clinical Immunology, Huddinge Hospital, S-141 86 Huddinge, Sweden. E-mail address: Back

3 Abbreviations used in this paper: IGHC, immunoglobulin heavy chain constant region; S, switch region; C, heavy chain constant region gene; PFGE, pulsed field gel electrophoresis. Back

Received for publication January 15, 1998. Accepted for publication June 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hofker, M. H., M. A. Walter, D. W. Cox. 1989. Complete physical map of the human immunoglobulin heavy chain constant region gene complex. Proc. Natl. Acad. Sci. USA 86:5567.[Abstract/Free Full Text]
  2. Lefranc, M. P., G. Lefranc, T. H. Rabbitts. 1982. Inherited deletion of immunoglobulin heavy chain constant region genes in normal human individuals. Nature 300:760.[Medline]
  3. Flanagan, J. G., T. H. Rabbitts. 1982. Arrangement of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing {gamma}, {epsilon} and {alpha} genes. Nature 300:709.[Medline]
  4. Rabbani, H., Q. Pan, N. Kondo, C. I. E. Smith, L. Hammarström. 1996. Duplications and deletions of the human IGHC locus: evolutionary implications. Immunogenetics 45:136.[Medline]
  5. Ghanem, N., M. P. Lefranc, G. Lefranc. 1988. Definition of the RFLP alleles in the human immunoglobulin IGHG gene locus. Eur. J. Immunol. 18:1059.[Medline]
  6. van der Zee, J. S., P. van Swieten, R. C. Aalberse. 1986. Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency. J. Immunol. 137:3566.[Abstract]
  7. Heiner, D. C., S. I. Lee, J. A. Short. 1986. IgG4 subclass deficiency syndromes. Monogr. Allergy 20:149.[Medline]
  8. Aalberse, R. C., R. van der Gaag, J. van Leeuwen. 1983. Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4 restricted response. J. Immunol. 130:722.[Abstract]
  9. Perelmutter, L.. 1984. IgG4: non-IgE mediated atopic disease. Ann. Allergy 52:64.[Medline]
  10. Djurup, R.. 1985. The subclass nature and clinical significance of the IgG antibody response in patients undergoing allergen-specific immunotherapy. Allergy 40:469.[Medline]
  11. Nikaido, T., Y. Yamawaki-Kataoka, T. Honjo. 1982. Nucleotide sequences of switch regions of immunoglobulin C{epsilon} and C{gamma} genes and their comparison. J. Biol. Chem. 257:7322.[Abstract/Free Full Text]
  12. Szurek, P., J. Petrini, W. Dunnick. 1985. Complete nucleotide sequence of the murine {gamma}3 switch region and analysis of switch recombination sites in two {gamma}3-expressing hybridomas. J. Immunol. 135:620.[Abstract]
  13. Petrini, J., B. Shell, M. Hummel, W. Dunnick. 1987. The immunoglobulin heavy chain switch: structural features of the {gamma}1 recombinant switch region. J. Immunol. 138:1940.[Abstract]
  14. Pan, Q., H. Rabbani, F. C. Mills, E. Severinson, L. Hammarström. 1997. Allotype-associated variation in the human {gamma}3 switch region as a basis for differences in IgG3 production. J. Immunol. 158:5849.[Abstract]
  15. Bottaro, A., M. de Marchi, G. G. de Lange, C. Boccazzi, L. Fubini, C. Borra, N. Cappello, A. O. Carbonara. 1989. Human IGHC locus restriction fragment length polymorphisms in IgG4 deficiency: evidence for a structural IGHC defect. Eur. J. Immunol. 89:2159.
  16. Olsson, P. G., L. Hammarström, D. W. Cox, C. I. E. Smith. 1992. Involvement of both HLA and Ig heavy chain haplotypes in human IgA deficiency. Immunogenetics 36:389.[Medline]
  17. Brusco, A., F. Cinque, S. Saviozzi, C. Boccazzi, M. de Marchi, A. O. Carbonara. 1997. The G4 gene is duplicated in 44% of IGHC haplotypes. Hum. Genet. 100:84.[Medline]
  18. Mattila, P., J. Korpela, T. Tenkanen, K. Pitkanen. 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase: an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19:4967.[Abstract/Free Full Text]
  19. Rabbani, H., N. Kondo, C. I. E. Smith, L. Hammarström. 1995. The influence of gene deletions and duplications within the IGHC locus on serum immunoglobulin subclass levels. Clin. Immunol. Immunopathol. 76:S214.[Medline]
  20. Hammarström, L., A. O. Carbonara, M. DeMarchi, G. Lefranc, M.-P. Lefranc, C. I. E. Smith. 1987. Generation of the antibody repertoire in individuals with multiple immunoglobulin heavy chain constant region gene deletions. Scand. J. Immunol. 25:189.[Medline]
  21. Mills, F. C., M. P. Mitchell, N. Harindranath, E. E. Max. 1995. Human immunoglobulin S{gamma} regions and their participation in sequential switching to IgE. J. Immunol. 155:3021.[Abstract]
  22. Wyatt, R. T., R. A. Rudders, A. Zelenetz, R. A. Delellis, T. G. Krontiris. 1992. Bcl-2 oncogene translocation is mediated by a chi-like consensus. J. Exp. Med. 175:1575.[Abstract/Free Full Text]
  23. Nilsson, L., P. Grant, I. Larsson, S. Pettersson, P. Sideras. 1995. The human I{alpha}1 region contains a TGF-ß1 responsive enhancer and a putative recombination hotspot. Int. Immunol. 7:1191.[Abstract/Free Full Text]
  24. Bottaro, A., M. de Marchi, N. Migone, A. O. Carbonara. 1989. Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus. Genomics 4:505.[Medline]
  25. Ghetie, V., E. S. Ward. 1997. FcRn: the MHC class I-related receptor that is more than an IgG transporter. Immunol. Today 18:592.[Medline]
  26. Ghanem, N., M. Bensmana, J. M. Dugoujon, J. Constans, M. P. Lefranc, G. Lefranc. 1989. BamHI and SacI RFLPs of the human immunoglobulin IGHG genes with reference to the Gm polymorphism in African people: evidence for a major polymorphism. Hum. Genet. 83:37.
  27. Bottaro, A., R. Gallina, M. DeMarchi, A. O. Carbonara. 1989. Genetic analysis of new restriction fragment length polymorphisms (RFLP) in the human IgH constant gene locus. Eur. J. Immunol. 19:2151.[Medline]
  28. Aoki, K., K. Nakahara, C. Ikegawa, M. Seto, T. Takahashi, J. Minowada, J. L. Strominger, R. T. Maziarz, M. Kasai. 1994. Nuclear proteins binding to a novel target sequence within the recombination hotspot regions of Bcl-2 and the immunoglobulin DH gene family. Oncogene 9:1109.[Medline]
  29. Smith, G. R.. 1983. Chi hotspots of generalized recombination. Cell 34:709.[Medline]
  30. Fujiwara, S., Y. Ono. 1995. Repetitive sequence in the Epstein-Barr virus EBNA-3C gene is related to a family of minisatellite arrays in the human genome. Virus Genes 11:31.[Medline]
  31. Kenter, A. L., B. K. Birshtein.. 1981. Chi, a promoter of generalized recombination in {lambda} phage, is present in immunoglobulin genes. Nature 293:402.[Medline]
  32. T., Morita, Y. Yoshimura, A. Yamamoto, K. Murata, M. Mori, H Yamamoto, A. Matsushiro. 1993. A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae Rad51 genes. Proc. Natl. Acad. Sci. USA 90:6577.[Abstract/Free Full Text]
  33. F. E., Benson, A. Stasiak, S. C. West.. 1994. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13:5764.[Medline]
  34. Li, M.-J., M.-C. Peakman, E. I. Golub, G. Reddy, D. C. Ward, C. M. Radding, N. Maizels. 1996. Rad51 expression and localization in B cells carrying out class switch recombination. Proc. Natl. Acad. Sci. USA 93:10222.[Abstract/Free Full Text]
  35. Tracy, R. B., J. K. Baumohl, S. C. Kowalczykowski. 1996. The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences. Genes Dev. 11:3423.[Abstract/Free Full Text]
  36. Rabbani, H., A. Bottaro, A. O. Carbonara, A. Brusco, U. Cariota, P. G. Olsson, C. I. E. Smith, L. Hammarström. 1993. The influence of gene duplications in the human immunoglobulin heavy chain constant locus on serum levels of immunoglobulin. Immunodeficiency 4:237.[Medline]
  37. Chen, C., B. K. Birshtein. 1997. Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C{alpha}1 and C{alpha}2 genes. J. Immunol. 159:1310.[Abstract]
  38. Mills, F. C., N. Harindranath, M. Mitchell, E. E. Max. 1997. Enhancer complexes homologous to a murine LCR lie downstream of both human C{alpha} genes. J. Exp. Med. 15:186.
  39. Pinaud, E., C. Aupetit, C. Chauveau, M. Cogne. 1997. Identification of an homolog of the C{alpha}3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream the human immunoglobulin {alpha}1 gene. Eur. J. Immunol. 27:2981.[Medline]
  40. Madisen, L., M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt’s lymphoma cells. Genes Dev. 8:2212.[Abstract/Free Full Text]
  41. Leung, H., N. Maizels. 1992. Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences. Proc. Natl. Acad. Sci. USA 89:4154.[Abstract/Free Full Text]
  42. Leung, H., N. Maizels. 1994. Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences. Mol. Cell. Biol. 14:1450.[Abstract/Free Full Text]
  43. Daniels, G. A., M. R. Lieber. 1995. Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences. Proc. Natl. Acad. Sci. USA 92:5625.[Abstract/Free Full Text]
  44. Li, M. J., W. Chung, N. Maizels. 1997. Developmental specificity of immunoglobulin heavy chain switch region recombination activities. Mol. Immunol. 34:201.[Medline]
  45. Ott, D. E., F. W. Alt, K. B. Marcu. 1987. Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells. EMBO J. 6:577.[Medline]
  46. Ott, D. E., K. B. Marcu. 1989. Molecular requirements for immunoglobulin heavy chain constant region gene switch-recombination revealed with switch-substrate retroviruses. Int. Immunol. 1:582.[Abstract/Free Full Text]
  47. Ballantyne, J., D. L. Henry, K. B. Marcu. 1997. Antibody class switch recombinase activity is B cell stage specific and functions stochastically in the absence of ‘targeted accessibility’ control. Int. Immunol. 9:963.[Abstract/Free Full Text]
  48. Dard, P., A. Sanchez-Mazas, J. M. Dugoujon, G. D. Lange, A. Langaney, M. P. Lefranc, G. Lefranc. 1996. DNA analysis of the immunoglobulin IGHG loci in a Mandenka population from eastern Senegal: correlation with Gm haplotypes and hypotheses for the evolution of the Ig CH region. Hum. Genet. 98:36.[Medline]
  49. Hassan, M. S., K. B. Islam, L. Hammarström, C. I. E. Smith. 1992. Regulation of C{gamma}3 expression: role of switch in the allotype-associated variation of human serum IgG3 levels. J. Immunol. 148:2555.[Abstract]
  50. Morell, A., F. Skvaril, A. G. Steinberg, E. van Loghem, W. D. Terry. 1972. Correlations between the concentrations of the four subclasses of IgG and Gm allotypes in normal human sera. J. Immunol. 108:195.[Abstract/Free Full Text]
  51. Pan, Q., Y. Lindersson, P. Sideras, L. Hammarström. 1997. Structural analysis of human {gamma}3 intervening regions and switch regions: implications for the low frequency of switching in IgG3 deficient patients. Eur. J. Immunol. 27:2920.[Medline]



This article has been cited by other articles:


Home page
 JEMHome page
L. Du, M. van der Burg, S. W. Popov, A. Kotnis, J. J.M. van Dongen, A. R. Gennery, and Q. Pan-Hammarstrom
Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination
J. Exp. Med., December 22, 2008; 205(13): 3031 - 3040.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
Q. Pan-Hammarstrom, A.-M. Jones, A. Lahdesmaki, W. Zhou, R. A. Gatti, L. Hammarstrom, A. R. Gennery, and M. R. Ehrenstein
Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells
J. Exp. Med., January 18, 2005; 201(2): 189 - 194.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pan, Q.
Right arrow Articles by Hammarström, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pan, Q.
Right arrow Articles by Hammarström, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS