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The Journal of Immunology, 2006, 176: 1655-1667.
Copyright © 2006 by The American Association of Immunologists

The Nucleotide Targets of Somatic Mutation and the Role of Selection in Immunoglobulin Heavy Chains of a Teleost Fish1

Feixue Yang*, Geoffrey C. Waldbieser{dagger} and Craig J. Lobb2,*

* Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216; and {dagger} United States Department of Agriculture, Catfish Genetics Research Unit, Thad Cochran National Warmwater Aquaculture Center, Stoneville, MS 38776


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mutations generally lead to nonbeneficial consequences. Yet in the humoral immune system, somatic mutation serves as a cornerstone of Ig and Ab diversity by modifying the H and L chain structures encoded by rearranged V(D)J segments. The postrearrangement modifications to Ig genes that include somatic mutation, as well as class-switch recombination and gene conversion, are dependent upon the enzyme activation-induced cytidine deaminase (AID)3 that converts deoxycytidine to deoxyuridine (1, 2, 3). Studies to determine the specific nucleotide targets of somatic mutation in mammals have identified the RGYW/WRCY motif (where R = A or G, Y = C or T, and W = T or A) as a principal hotspot for AID-induced G:U lesions (4, 5, 6). In addition, the dinucleotide target WA has been identified as a principle site for A/T mutations (7, 8, 9, 10, 11, 12), which in turn has led to studies to define the roles of the error-prone polymerases in mismatch repair and their potential involvement as secondary mutators (13, 14, 15, 16, 17, 18, 19).

Somatic mutation in mammals is intimately involved in the co-related processes of B cell selection by Ag, which occurs in germinal centers, and affinity maturation. In this developmental pathway, mutated B cells with higher affinity receptors outcompete other B cells for limited amounts of Ag and clonally proliferate, whereas B cells with lower affinity receptors presumably undergo apoptosis. This process results in the production of Ab populations with higher affinity sites for Ag, which progressively increase in time (20, 21). There appears to be at least one other pathway where B cell maturation likely exists. This pathway is located in the splenic marginal zone wherein somatic mutation results in highly mutated IgM B cells that are involved in the T-independent response to Ags. Although it is not yet known whether selection by Ag results in affinity maturation within this IgM B cell population, it is known that affinity maturation can occur in the absence of germinal center formation (22, 23, 24, 25, 26, 27).

In contrast to the extensive studies of somatic mutation done in mammals, few studies have addressed the early evolutionary processes of somatic mutation and the nucleotides that are targeted for mutation. Earlier studies in xenopus and shark H chains indicated that somatic mutation occurred in these two classes of vertebrates and that there was a strong mutational bias toward G and C (28, 29). Subsequent studies on shark L chains and shark NAR have shown that mutations in A and T can account for 40–50% of the mutations. These latter studies have also observed that tandem mutations, ranging in length from 2 to 4 nts, can represent from 25 to 50% of the total mutations, suggesting that alternative mutational and/or repair mechanisms may exist (30, 31).

At present, there have been no definitive studies that prove whether or not somatic mutation occurs in the Igs of bony fish (class Osteichythes). Earlier studies in the channel catfish have defined 13 different VH families that are used in the H chain cDNA repertoire, and germline segments representing each family have been identified (32, 33, 34). The DH locus, identified through approaches that examined the excision productions of DH-JH recombination events, comprises at least three DH segments and is located ~9 kb upstream of the nine segments that compose the JH locus (35, 36, 37). During repertoire analyses, we observed that point mutations occurred in the JH-encoded region of H chain cDNA, and these initial observations have led to this report, which shows that somatic mutation occurs within catfish H chain V regions. We have subsequently analyzed the mono-, di-, and trinucleotide mutational targets as well as the occurrence of mutations within RGYW/WRCY motifs. These studies, coupled with analyses to determine whether there are selection mechanisms, provide new insight into the early evolutionary patterns and role of somatic mutation in Ig diversification.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Construction of library and sequence analysis

An Ig H chain cDNA library was constructed using total RNA from the spleen of an individual catfish (Ictalurus punctatus) as reported earlier (34). Briefly, first-strand synthesis was initiated with a primer corresponding to the Cµ2 domain of the catfish H chain, the product was tailed, and 30 rounds of PCR amplification using Taq polymerase (Invitrogen Life Technologies) were conducted using a primer for the Cµ1 domain and the adapter primer provided in the 5'-RACE kit. The amplicons were ligated and cloned into the T/A cloning vector pCR2.1, and individual colonies were transferred to master plates for subsequent analyses. From this library, 187 cDNA clones (gene accession nos. DQ230539–DQ230706, and sequences previously reported; Ref.34) representing rearranged members of the VH1 to VH13 gene families were defined by hybridization and sequenced with vector primers using ABI PRISM BigDye Terminators chemistry on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) in the U.S. Department of Agriculture, Agricultural Research Service, Mid-South Area Genomic Laboratory. FR and CDR regions were assigned using the nomenclature of the international ImMunoGeneTics information system (38).

In the 104 sequences that used a known germline VH gene or one of the defined VH consensus sequences (see Results), clones were identified that represented the same VDJ rearrangement and thus were deemed members of the same clonal set. The number of clones within a clonal set ranged in size from 2 to 9, and identical as well as different nucleotide mutations were observed. Because a mutation could be carried during clonal expansion, a mutation observed in the same position in a clonal set was deemed to represent a single event and therefore was only counted once in mutational analyses. In the VH analyses of these 104 clones, there were 388 mismatches identified in 39,083 total nucleotides. Similarly, the JH-encoded region within all 187 clones were aligned with the nine germline sequences designated JH1–JH9 defined in earlier studies (36), and mismatches with the assigned germline segment were recorded. There were 79 mismatches identified in 9,129 total nucleotides with identical mismatches within members of clonal sets counted as a single event. Each of the mutations identified in this study were manually verified by inspection of the sequence chromatograph, and the CS designation following the name of the clone was added to sequences assigned to clonal sets.

Determination of Taq polymerase fidelity

A rearranged H chain cDNA clone that used a member of the VH6 family (VH6VDJ) was amplified using the same PCR conditions that were used for cDNA library construction with the exception that the clone was subjected to 30 x 3 cycles (90 rounds) of amplification. The product was cloned into the pCR2.1 vector. Clones were subsequently sequenced, and 21 mismatches were identified in the 7,714 bases. The resultant Taq polymerase error rate was 0.30 x 10–4 mutations/bp per cycle. Therefore, Taq polymerase misincorporation errors within the VH-encoded region should represent no more than 0.32 to 0.36 mutations per sequence.

Calculation of mutability indexes

The mono-, di-, and trinucleotides compositions of the utilized VH and JH regions of the germline or consensus sequences were determined using Pustell software (IBI) and adjusted manually. The number of mutations in each sequence was recorded in an Excel spreadsheet. Mutability indexes were calculated as reported by Shapiro et al. (11) and are defined as the observed number of times a given mono-, di-, or trinucleotide target was mutated divided by the expected number of mutations. The frequency of a target in the database was initially determined as the total number of times a specific target existed in the database divided by the total number of all potential targets within the database. This frequency value was then multiplied by the total number of mutational events to yield the expected number of mutations.

Mutability indexes for each position in di- and trinucleotides targets were calculated separately. Thus, for dinucleotides, each mutation was counted twice (position 1 and position 2). In the trinucleotide database, every mutation was counted three times. In contrast, in the analysis of codons (extending from FR1 through the end of FR3) the mutations were only counted in a single position.

Statistical analysis

{chi}2 analysis was used to compare the mutational events in mono-, di-, trinucleotide, and codon analyses by contrasting the observed mutational frequencies to their expected mutational frequencies. p values <0.01 were considered statistically significant, and Bonferroni corrections were applied when the distribution of mutations resulting from a single mutation could be assigned to different di- or trinucleotide targets. {chi}2 tests were also performed in the analyses of mutational frequencies and distributions of various motifs within FR or CDR as reported within the Results. Fisher’s exact test was used to confirm the significance of the {chi}2 test when the expected counts of 25% of the cells had values <5 in the comparisons of Taq polymerase error rates. Wilson confidence intervals for binomial parameters were calculated to confirm the significance interval of the {chi}2 tests in the analyses of specific mutated positions within codons and in RGYW/WRCY targets. Statistical analyses of Ag selection pressure on Ig genes used the multinomial distribution model of Lossos et al. (39), which the authors have made available online at <http://www-stat.stanford.edu/immunoglobulin/>. The excess of CDR replacements or the scarcity of FR replacements were judged significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Somatic hypermutation occurs within both JH- and VH-encoded regions of catfish H chains

An Ig H chain-specific cDNA library constructed from the spleen of an adult channel catfish was screened with different VH family specific probes. From this library, 187 nonidentical clones representing various expressed members of the 13 different catfish VH families were identified. The JH-encoded region in these clones were aligned with the genomic sequences of the previously defined JH segments (designated JH1–JH9; Ref.36), and the germline JH segment used in the rearrangement was determined. These alignments showed two important features. The first was that nucleotide mismatches were observed in the expressed JH segment when compared with the sequence of the used germline JH. These mismatches could not be explained by potential allelic variation because the library was constructed from the cDNA from only one animal. Secondly, clones could be assigned to clonal sets that were defined as sequences that used the same VDJ rearrangement. Within a clonal set, identical mismatches from the germline JH sequence were observed in some but not all of the clones within a set. Because it was possible that somatic mutation occurred within the JH-encoded region, and since it was also possible that mutations could be maintained during B cell clonal expansion, a mutation in the same position in members of clonal sets was deemed to represent a single event and therefore only counted as a single mismatch. By these criteria, there were a total of 79 nucleotide mismatches in the cDNA clones when compared with the sequences of the germline JH segments (Fig. 1). These results indicated that these differences were due to either somatic mutation or to Taq polymerase errors that arose during PCR amplification.


Figure 1
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  		FIGURE 1. The germline coding region sequences of channel catfish JH segments and the locations where somatic mutations occurred within the JH-encoded regions of splenic H chain cDNA clones. The nucleotides introduced into the JH-encoded regions by mutation are shown directly underneath each JH germline sequence (designated JH1–JH9; Ref.36 ). The sequence of JH5 is not shown because no mutations were identified in the cDNA clones that used this segment. The coding regions of the JH3 and JH4 germline segments are identical except at their 5' ends, and deletion of these characteristic nucleotides during rearrangement results in cDNA clones that could have used either segment; such clones are designated as having used JH3/4. The demarcation of the FR4-encoded region is shown.

 
To determine whether these mismatches could be explained by Taq polymerase misincorporation errors, a single H chain cDNA clone (VH6VDJ) was arbitrarily selected and extensively amplified by PCR. The resulting PCR products were cloned, and representative products were sequenced. These results identified 21 nt substitutions within the sequenced 7,714 bases that were attributable to Taq misincorporation errors. Because these differences could not be assigned to a specific strand, the 12 possible substitutions were grouped into their respective six transition/transversions categories and the distribution of the errors compared with that reported in the literature (Table I). Our control Taq polymerase error rate was 0.30 x 10–4 errors/bp per cycle, which was consistent with the Taq error rate found by others (reviewed in Ref.41). The comparison of the distribution of these misincorporations was also not statistically different from reported literature values (Fisher’s exact text, p = 0.74; Table I). Substitutions for A or T were more abundant than substitutions for G or C, and transitions accounted for 81% of the Taq-attributed differences. In contrast, the distribution of the JH substitutions was statistically different from our internal control (VH6VDJ) as well as the literature-reported Taq polymerase error rate (Fisher’s exact test, p < 0.0001; Table I). G or C transversions, infrequent in control misincorporations, accounted for 16.5% of the substitutions in JH-encoded regions.


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  		Table I. Distribution of nucleotide substitutions resulting from Taq polymerase errors compared to the distribution of substitutions identified in JH- and VH-encoded regions of channel catfish H chain cDNA clones

 
With these studies indicating that somatic mutation occurred in JH-encoded regions, the VH-encoded regions within the 187 clones were assigned to their respective VH family, and the sequences were aligned. Of the 187 sequences, 104 of these could be readily assigned to 11 different groups. The VH-encoded regions within each group shared between 95 and 100% nucleotide identity and exhibited coding region similarities likely restricted to a specific VH germline gene (32, 33, 34). Nucleotide differences between aligned clones within a group were principally single position differences, and these infrequent differences were located at various positions within the VH-encoded region. Six of these 11 groups also contained multiple cDNA clones that shared the same VDJ rearrangement and were therefore members of clonal sets (Table II). These results allowed us to construct a VH consensus sequence for each group that was derived from clones that represented three or more independent VDJ rearrangements. The VH consensus sequence generally shared >98% nucleotide identity to the VH-encoded region of each cDNA clone assigned to that group (Table II).


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  		Table II. Summary of the mutations in cDNA sequences assigned to 11 VH consensus sequences

 
When the consensus sequences were aligned with the respective sequences in these 11 groups, there were a total of 388 mismatches in the 39,083 VH-encoded nucleotides (nucleotide differences observed in the same position in members of clonal sets were recorded only once). The distribution of these VH substitutions was significantly different from the distribution of misincorporation errors induced by Taq polymerase when compared with the literature data or to our internal control (p < 0.0001; Table I). There was, however, no significant difference between the distribution of the substitutions within the VH- and JH-encoded regions (p = 0.23; Table I). We therefore conclude that somatic mutation occurred within catfish H chain V regions.

Specific nucleotides are differentially targeted by somatic mutation

With these results, it was important to determine whether specific bases were preferentially targeted in VH coding regions by somatic mutation and to compare the pattern of these mutations with those defined in mammals. Mutability indexes, defined as the observed frequency of the targeted nucleotide compared with its expected unbiased mutation frequency, were determined (7, 11). Mutability indexes were normalized to take into consideration the fact that a specific nucleotide may not occur at the same relative frequency as the other three (7). These analyses showed that G and C were preferentially mutated (p < 0.001 and p < 0.005, respectively), and that the mutability indexes for these two nucleotides were similar (Table III). G and C mutations were significantly higher than the mutations that occurred in either A or T (p < 0.001), and mutations in T were significantly lower than mutations in A (p < 0.005).


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  		Table III. Substitutions and mononucleotide mutability indexes in the VH-encoded regions of channel catfish IgH chains

 
A total of 60.3% of the substitutions were transitions, with R transitions 19% higher than Y transitions. R transversions (62.3%) were also more common than Y transversions. The overall transversion to transition ratio was 0.66:1. This ratio is significantly different from the theoretical 2:1 transversion to transition ratio (p < 0.001). The transversion:transition ratios for A, C, G, and T were 0.75, 0.61, 0.76, and 0.43, respectively, and each was also statistically different from the theoretical ratio (p < 0.001). Thus, it appears that neither the targeting of mutations nor the patterns of substitutions that subsequently occur in those positions can be explained by assuming that somatic mutation events occur randomly.

Sequence-specific patterns of somatic mutation

Mutation hotspots have been identified in mammals that represent sequence-specific patterns where the mutation rate of a target is influenced by the presence of specific neighboring bases. To determine whether specific dinucleotides had higher mutation frequencies in the VH database, mutability indexes were calculated for mutations in the first position, the second position, or the combined positions (position independent) for each of the possible 16 dinucleotides (Table IV). The significantly mutable dinucleotides by position were CT, its reverse complement AG, and GC (where the underlined nucleotide(s) indicates the significantly mutated position). The only dinucleotides that were significant mutable in the combined positions were AG and GC. Dinucleotide mutability indexes for the mutations that occurred within the JH-encoded regions were also determined. Although the number of these mutations did not permit statistical evaluation by dinucleotide position, {chi}2 analyses of the combined positions showed that only AG and GC were significantly mutable (p < 0.01).


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  		Table IV. Comparison of dinucleotide mutability indexes in channel catfish and human VH regions

 
Shapiro et al. (11) had determined dinucleotide mutability indexes for human VH somatic mutation events. The comparison of this data with the present analyses indicates that AG and GC were significant targets of mutation in both humans and catfish VH regions (Table IV). Both of these targets as well as the CT are located within the mutation hotspot RGYW/WRCY. In the human VH analyses, the additional mutation targets of AT, AA, and TA were also identified. The latter two targets compose the WA motif (where W = T or A), which has been reported as an additional target for mammalian somatic mutation events (7, 8, 9, 10, 11, 12, 13). The WA motif is also represented in WRCY motifs (although the targeted nucleotide is not in the C position). None of these WA dinucleotide motifs, however, were significant targets of mutation in catfish VH regions (Table IV).

To determine whether the mutations in catfish VH regions might reflect alternatives to the hotspot motifs characterized in mammals, trinucleotide mutability indexes were calculated for each VH mutation in each of the three possible positions. In addition, the position-independent trinucleotide mutability indexes (shown as "combined" in Table V) was determined. These indexes were compared with the trinucleotide mutability indexes derived by Shapiro et al. (11, 43) for human VH mutations (Table V). These comparisons indicate several important points. First, the number of VH trinucleotide mutation targets in catfish is restricted when compared with those that occur in man. In the analysis of the combined positions there were five significantly mutable trinucleotide targets in catfish VH, whereas 13 such targets were identified in the human VH studies. In the analysis of the mutations by position, nine were identified in catfish, and 29 targets were identified in the human VH studies. Secondly, 11 of the 14 total mutation targets identified in catfish were also present in the human VH studies. Among these is AGC and GCT; both of these are major targets of somatic mutation in both species, and both are contained within the RGYW/WRCY motif. In addition, it is apparent that there is a distinction between the WAN motifs targeted in man compared with the catfish. TAN, and to a lesser degree AAN, are both significantly mutable in man, but none of these motifs was significantly mutable in the catfish database. Therefore, WAN is not a preferred target for somatic mutation events in the catfish.


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  		Table V. Comparison of trinucleotide mutability indexes in channel catfish and human VH regions

 
In addition to the trinucleotides AGC and GCT, significantly mutable trinucleotides in the first position included CTA, CTC, and GCA. These motifs, however, may not be additional targets for mutation because the first two nucleotides of CTA and CTC are in the WRCY motif, and GCA is found in the RGYW motif. The other two significantly mutable trinucleotides were AAG and TAG. These may also not represent novel target motifs because AG is a significantly mutable dinucleotide and represents the first two nucleotides contained in RGYW. The latter trinucleotides may simply represent major contributors to a NAG motif considering the relatively high (although not significant) mutability indexes of CAG and GAG.

Somatic mutations are restricted to specific sequences within RGYW/WRCY motifs

The above di- and trinucleotide analyses did not identify any significant motifs with G in the R position of RGYW (i.e., GG, NGG, GGC, GGT) or any significant motifs with C in the Y position of WRCY (i.e., CC, GCC, ACC). These results indicated that somatic mutation events in catfish may have restricted targets, and we proceeded to determine the occurrence and patterns of mutations in these motifs. RGYW/WRCY motifs represented 28.7% of the 39,083 nucleotides in the VH database, and these motifs were significantly overrepresented when compared with their expected distribution (p < 0.0001). These general motifs accounted for 183 of the total mutations in the VH database (47.2%) and were statistically significant targets of mutation events (p < 0.001). The number of the mutations in each of these motifs was then determined to address which of these motifs were targets for somatic mutation (Table VI). These results showed that of these 15 different motifs only AGCT, AGCA had significant position-independent mutability indexes (p < 0.01).


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  		Table VI. The occurrence and mutability indexes of the RGYW and WRCY motifs in catfish VH-encoded regions

 
The specific mutations that occurred within the RGYW/WRCY motifs were then determined. Fifty-three of the 118 total VH mutations that occurred in G were located in RGYW motifs (44.9%), and 43 of the 108 total mutations that occurred in C were located in WRCY motifs (39.8%). Mutations in these positions were significantly higher than the overall mutation rate of G and C in the VH database (p < 0.001). However, when the mutability indexes of G and C were determined in each of these motifs, only AGCT and AGCA were significant targets for G mutations in the RGYW motifs, and only AGCT was a significant target for C mutations in the WRCY motifs (Table VI). The other RGYW motifs accounted for nine G mutations, and the other WRCY motifs accounted for 16 C mutations; none of these motifs were significant hotspots for G and C mutations (p > 0.01).

AGCA had the second highest position-independent mutation frequency in these analyses, but only 19 of the 42 mutations occurred in the G position. When the specific mutations in the AGCA motif were examined, 10 mutations occurred in the C position, and this motif was determined to be a significant hotspot for C mutations (p < 0.01). Therefore, the motif that best describes G and C hotspots in the catfish VH database is AGCW. This motif explains 37.3% of the mutations that occurred in G and 33.3% of the mutations that occurred in C. We tested this conclusion by removing the AGCW motifs from the VH database, and then we reanalyzed the remaining mutations for significant trinucleotide mutability indexes. {chi}2 analyses could only be conducted with confidence on the mutations in the combined positions of a trinucleotide (position independent), and these results showed that none of the resulting trinucleotides were significantly mutable (p > 0.01). Lastly, we examined the flanking A nucleotides in AGCA to determine whether either of these positions had significant incidence of mutations. Statistical analyses showed that AGCA, which had nine mutations, was a significant hotspot for A mutations (p < 0.01). Therefore, we conclude that AGCW is a significant target for G and C mutations, and AGCA is a significant target for A mutations.

Patterns of somatic mutations in codons

Three hundred thirty-one of the 388 mutations in the VH database were located within the region spanned by FR1 through the end of FR3; 251 mutations were located with the FR regions, and 80 mutations were within the CDR regions. The overall mutability index of the codons within the three combined FR regions (FR1, FR2, and FR3; designated as FRT) was 0.90, whereas the overall mutability index of codons within the combined CDR regions (CDR1 and CDR2, designated CDRT) was 1.50. {chi}2 analyses showed that codons in CDRT were significantly more mutable than those found in FRT (p < 0.001). Mutability indexes for the individual codons were then derived and their statistical significance evaluated. Position-independent mutational analyses showed that only three codons were significantly mutable targets: AGC, GCA, and GCT (p < 0.01).

Mutations in AGC codons accounted for 34 of the 80 mutations found within CDRT (42.5%), but only 16 of the 251 mutations found in the FRT (6.4%). These values indicated that either AGC codons were significantly more mutable in CDR regions and/or that the distribution of AGC codons between the FR and CDR regions was different. To test this hypothesis, we analyzed the distribution of the serine codons AGC, AGT, and TCN within the FRT and CDRT of the VH consensus sequences. Serine codons represented 10.8 and 23.1% of the FRT and CDRT codons, respectively, and the ratios of AGC:AGT:TCN were 1:0.75:4.18 in FR regions and 1:0.42:0.63 in CDR regions. {chi}2 analyses showed that AGC was significantly more represented in CDR regions than in FR regions (p < 0.001). We then compared the mutation frequency of the AGC codon in the FRT and CDRT. In the VH database AGC represented 301 codons, with 135 located in FRT and 166 located in CDRT; the number of mutated AGC codons in these regions was 16 and 34, respectively. {chi}2 analyses indicated that the AGC codon in CDRT was not significantly more mutable than that found in FRT (p = 0.045). Comparisons with the other highly mutable codons GCT and GCA showed that neither of these codons was more frequently represented in CDRT than FRT, and only GCA, which accounted for seven of the mutations in CDRT, was more highly mutated in CDRT than in FRT (p < 0.001). Thus, these results indicate that the nonrandom distribution of the highly mutable AGC codon within CDRT appears to primarily explain the higher overall CDR mutation rate.

The impact of selection on somatic mutations

In mammalian productive rearrangements, selection influences the patterns of mutation because certain amino acid positions do not appear to tolerate replacements, whereas mutations in other positions appear to occur as the result of selection by Ag. To initially address the question of selection in catfish productive rearrangements, we analyzed the positional distribution of the 331 mutations within the codons in the FR1 through FR3 VH-encoded region. There were a total of 104 mutations in codon position 1, 110 mutations in codon position 2, and 117 mutations in codon position 3. There was no significant difference in the distribution of mutations in these positions (p = 0.68), and this was also true when the positions of mutations in codons located only within FRT were examined (FRT, p = 0.82). The positions of the mutations in codons within the CDRT were also not significantly different if the AGC codon was removed (p = 0.34).

Of the 331 total mutations within the FR1 to FR3 regions, 251 mutations were in FRT resulting in 173 replacement (R) substitutions and 78 silent (S) substitutions, whereas 80 mutations were located in CDRT resulting in 51 replacement substitutions and 29 silent substitutions. {chi}2 analyses showed that there was no significant difference in the R:S ratios when the FRT and CDRT regions were compared (p = 0.39). We also analyzed the substitutions that occurred only in the AGC codon. In FRT, there were 16 mutations resulting in 11 replacement and 5 silent substitutions; in CDRT, there were 34 mutations resulting in 22 replacement and 12 substitutions. {chi}2 analyses also showed that there was no significant difference between these R:S ratios (p = 0.78). Although these combined results suggest the lack of selection (10, 44), the work of others has shown that analyses must also focus on the individual sequences rather than the collective data. Lossos et al. (39), building on the earlier work of Chang and Casali (45), developed a multinomial distribution model to estimate Ag selection pressure on expressed Ig genes. In this model, selection is addressed by determining the excess of replacements in CDR and/or the scarcity of replacements in FR. p values are derived by determining the number of replacement and silent substitutions, and values with significance p < 0.05 are assumed to have resulted by selection rather than by chance. We compared each of the 93 catfish productive rearrangements that had mutations within the FR1 through FR3 region with their respective VH consensus sequence using the Lossos et al. (39) distribution model. In 82 of these sequences (88%) there was no statistical evidence to suggest either selection alternatives (i.e., excess of CDR replacements or scarcity of FR replacements). In 10 of these rearrangements, there was counterselection of mutations in FR because these exhibited significant scarcity of FR replacements. Four of these rearrangements are in clonal set VH7B-CS2 (see below). Only 1 clone exhibited significant excess of CDR replacements, and none of the rearrangements exhibited both significant scarcity of FR replacements and excess CDR replacements.

To further determine whether selection mechanisms may be present, we analyzed the patterns of somatic mutations in three clonal sets representing different VDJ rearrangements. These sets were chosen because they had at least seven different clonal representatives to identify the unique and sequential mutations that likely occurred during clonal expansion. For each of these sets, the progenitor VDJ sequence was defined using the VH member consensus sequence for 5'-untranslated region through FR3, the clonal set consensus sequence for CDR3 with the germline sequence of the utilized region of the DH segment, and the sequence of the utilized germline JH segment for FR4. Different basic patterns of clonal genealogies were evident in each of these three sets (Fig. 2). The first, depicted by clonal lineage set VH10A-CS1, showed that clone 2G03 had four mutations when compared with the progenitor consensus sequence. Each of the other clones within the set likely descended from 2G03, and these clones had accumulated one to three different mutations that were not present in 2G03 or in each other. In clonal set VH9A-CS1 a different pattern was observed in that five of the clones (2H10, 2B08, 2C03, 2F03, and 2E04) represented different lineages that descended from the progenitor VDJ consensus. These radiating clonal descendents had accumulated from one to three mutations, and the mutations in one sublineage were different from those found in other sublineages. Two other clones (2C06 and 2F11) had descended from a hypothetical intermediate, designated H1 (Fig. 2B), which had accumulated four mutations since it had descended from the parental VDJ. Clone 2C06 had accumulated four additional mutations, whereas 2F11 had a single additional mutation in comparison to H1.


Figure 2
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  		FIGURE 2. Genealogical relationships between clonally related splenic cDNA sequences. A–C, Three different clonal sets that used members of three different VH families (VH10, VH9, and VH7, respectively) are shown. Within each clonal set, the dashed circle indicates the common progenitor, the dotted circle(s) represents the hypothetical (H) intermediate(s), and the solid circle represents the sequenced cDNA clone designated by its name. The number besides each arrow indicates the number of different mutations introduced during clonal expansion. For example, in A, clone VH10A2G03-CS1 had accumulated a total of four different mutations since it had descended from its progenitor, and each of these four mutations were found in all other clonal descendents. Each of the other descendant clones had in turn accumulated additional mutations as indicated by the number adjacent to the arrow. D, The multiple sequence alignment of the cDNA clones within clonal set VH7B-CS2. The sequence of the progenitor consensus (7B-Cons) is shown on the top line and is demarcated into the 5'-untranslated region, FR and CDR regions, and partial Cµ region. The utilized regions of the germline sequence of the DH3 segment (overlined; Ref.37 ) and the germline JH2 segment (underlined; Ref.36 ) are shown. Dots indicate sequence identity with the 7B-Cons sequence, and dashes indicated nucleotides that were absent from the 5'- ends of clones 1E10 and 6H01; the nucleotides introduced by mutation are indicated.

 
The third clonal set, VH7B-CS2, was different from each of these other clonal sets. Various major lineages appear to have descended from the progenitor VH7B VDJ sequence, and the mutations that accumulated in the clonal descendants were extensive. Lineage analysis required that two hypothetical intermediates, designated H1 and H2, be constructed to serve as clonal intermediates in clonal expansion. H1 was postulated because clones 6E07 and 6D07 had mutations in common, and therefore a common intermediate was likely. These two descendant clones had extensively diverged from each other with a total of 27 different mutations between them. All of the other clones within this clonal set were derived from a different sublineage. This sublineage had accumulated eight common mutations when compared with the progenitor, and these identical mutations were presumed to have been present in an intermediate designated as H2. Two subsequent pathways had occurred within this sublineage. The first, represented by clone 6A02, had accumulated an additional 13 mutations from H2. The second, represented by the other remaining clones, descended from a common intermediate (1E10), which itself had accumulated four additional mutations in comparison with H2. Thus, the mutations present within this clonal set are extensive, with the most distant descendant in this set (6A02) having diverged from its progenitor by 21 mutations.

To determine whether selection by Ag had occurred within any of these three clonal sets, the multinomial distribution analyses of replacement and silent substitutions extending through the end of FR4 were calculated. These results showed that when the members in these clonal sets were compared with either their progenitor or to their immediate clonal precursor (as shown in Fig. 2), only 1 of these clones had significant p values for either scarcity of FR replacements or excess CDR replacements. Clone 3B08 in VH7B-CS2, which had 17 total mutations compared with its progenitor, was marginally significant for scarcity of FR replacements (p = 0.044). Thus, within these clonal sets selective forces to either conserve FR or to accumulate R mutations in CDR do not appear to exceed that expected to occur by chance.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies have sought to provide insight into the nucleotide targets and potential mechanisms of somatic hypermutation that are operational at the phylogenetic level of bony fish. The analyses of mononucleotide mutations indicated that A, C, G, and T mutations accounted for a relative mutation rate in catfish VH regions of 27, 28, 30, and 15%, respectively, with transitions more frequent that transversions. When corrected for base composition, mutations in A, C, G, and T nucleotides occurred with mutational frequencies of 23, 31, 33, and 14%, respectively. Mutability indexes were determined, and statistical analyses showed that the number of mutations in A were not significantly different from that expected from sequence-insensitive (random) mutations. C and G were mutated at frequencies higher than expected from random mutations, and mutations in T were significantly lower than expected. Thus, the mutational frequencies of G and C (unlike A and T) are approximately equal. G and C are the preferred targets for somatic mutation, and there is a bias against mutations occurring in T.

These results can be compared with studies done in other vertebrates. For example, Milstein et al. (8) reported average mutational frequencies of A, C, G, and T of 33, 23, 24, and 20%, respectively, for human and mouse H and L chains and artificial substrates inserted into murine transgenes. Smith et al. (7) reported mutation frequencies of 33, 26, 24, and 16%, respectively, in a collective study on mutations in murine V genes. There was no significant difference when these frequencies were compared with the mononucleotide mutation frequencies defined in catfish VH or JH databases (p > 0.1). The generally observed imbalance of A compared with T mutations in mammalian systems has been a basis for proposing a strand-biased mechanism that differentially targets A:T but not G:C pairs (discussed below). Earlier studies on shark (29) and Xenopus (28) H chains had analyzed a limited number of mutations and concluded that these lower vertebrates exhibited a strong mutational bias toward G and C. However, extensive mutational analysis on shark L chains (30) and shark new Ag receptor (31) have subsequently shown that mutations in A and T generally represent >40 and >50% of the mutations, respectively. These latter studies have also observed that tandem mutations, ranging in length from two to four nucleotides, are characteristic of the mutational pattern and may represent >50% of the total mutations. The percentage of transitions also varied when substitutions in point mutations were compared with those observed in tandem mutations. These results have suggested that alternative mutational and/or repair mechanisms may be operational. Tandem mutations, however, are not characteristic of the mutational pattern in catfish H chains as these represent <5% of the total mutations.

The analyses to determine the nucleotide targets of somatic mutation in the catfish have shown that specific motifs target specific nucleotides for mutation. Dinucleotide analyses of the VH and JH databases showed that CT, its reverse complement AG, and GC are significant targets for G and C mutations. No significant dinucleotides were identified that were targets for either A or T mutations in either database. The lack of A and T dinucleotide targets suggested that trinucleotide analyses might define targets for A and T mutations if these mutations occurred in highly specific targets. In addition, these analyses would determine whether G and C mutations were more restricted than the dinucleotide analyses had indicated. Trinucleotide analyses were conducted on the mutations in the VH database, because the JH database had insufficient inherent structural diversity. Although none of the trinucleotides were identified as significant targets for A or T mutations, nine significantly mutable positions in seven different trinucleotides were identified as significant targets for G or C mutations. These seven motifs were AAG, AGC, CTA, CTC, GCA, GCT, and TAG. The two palindrome motifs AGC and GCT, respectively, accounted for 39 or 29% of the total mutations in G and accounted for 38 or 34% of the total mutations in C. The other highly mutable C trinucleotide targets (CTA and CTC) accounted for a combined total of 35% of the C mutations. Of the remaining three trinucleotides (GCA, AAG, and TAG) GCA was the most mutated and accounted for 21% of the G mutations; the other two motifs accounted for 14 and 8% of the G mutations, respectively. It must be noted that these separate percentages should not be considered as additive. For example, a single mutation that occurred in the G position of the tetranucleotide AGCT would be counted in both AGC and GCT.

The vertical alignment of these targeted trinucleotides indicated that many of these motifs might be included in RGYW/WRCY motifs, and we proceeded to test this hypothesis. RGYW/WRCY motifs were found to be significantly overrepresented in the VH database; nonetheless, these motifs were significant targets of mutations accounting for 47% of the total VH position-independent mutations. Position-dependent analyses, however, showed that only two of these general motifs were significant targets for G and C mutations, AGCT and AGCA. These two combined motifs explained 37% of the total mutations in G and 34% of the total mutations in C. In addition, these analyses showed that AGCA was a significantly mutable target and accounted for 9% of the total mutations that occurred in A. These results were tested by removing the AGCW motifs from the VH database, and the mutability indexes of the resulting trinucleotides were recalculated. These results showed that none of these resulting trinucleotides were now significantly mutable (p > 0.01). Thus, we conclude that these three motifs (AGCT, AGCA, and AGCA) are the principal motifs for specifically targeted somatic mutations in catfish H chains.

These results allow comparisons to be made with the somatic mutations events characterized in mammals. Two general features of somatic mutation events in mammals have become evident. The first is that approximately equal numbers of mutations appear to occur in G and C nucleotides. The second is that the number of mutations in A generally exceeds the number of mutations in T (7, 8, 46, 47). These results have been one of the foundations for proposing two underlying mechanisms or stages for somatic mutation. The first targets G and C nucleotides and is strand independent. It is now known that mutations in G and C are principally due to AID, which catalyzes deamination of C residues to U residues and preferentially targets RGYW/WRCY motifs (1, 2, 5). The DNA deamination model predicts that when the lesion in G:U pairs is repaired, faithful replication would convert the U to a T and result in the observation that transitions predominate at the mutated sites (48). These features of mutational analyses in mammals also appear to be characteristic of the mutational patterns observed in catfish H chains (Table III). In regards to the specific targeting of mutations to RGYW motifs, it is clear that the spectrum of RGYW targets used in mammals is restricted in catfish. Dorner et al. (49) calculated the position-independent number of mutated RGYW motifs in productively rearranged human H chains (mutations in WRCY were not reported). In these analyses, 28.2% of the total mutations were located in RGYW motifs with 13.2% of the total mutations located in AGCW. In the catfish database, 35.6% of the total mutations were located in RGYW motifs with 25.5% of the total mutations located in AGCW. Therefore, the targeting of mutations to RGYW motifs as well as the number of mutations targeted to AGCW is significantly higher in catfish H chains ({chi}2, p < 0.01; also see mutability indexes of RGYW/WRCY motifs; Table VI).

In contrast to G and C mutations, the imbalance of mutations in A and T has indicated that there is a strand-biased mechanism for mutations in A/T that preferentially occurs in the WA motif (9, 50, 51). Extensive studies to explain these observations have implicated numerous repair enzymes involved in resolving U:G mismatches (recently reviewed in Refs.14, 15, 16, 17, 18, 19). Although discussion of these enzymes and mechanisms is beyond the scope of this paper, no evidence was obtained in these studies to indicate that the WA motif is a targeted site for mutation in catfish V regions. However, the conclusion that AGCA is a hotspot for A mutations supports the hypothesis that resolution of U:G mismatches in this RGYW motif involves mutations in adjacent sites. Following the recent conclusions of Neuberger et al. (14), this may be the first report to provide direct linkage of significant mutations in C:G pairs and adjacent A:T pairs. This result was detected in these studies because of the high concentration of mutations targeted toward limited RGYW motifs. If it is assumed that G:C mutations in catfish are targeted by AID, then a two-step or second stage of mutation, which uses an independent mutational mechanism targeted toward A:T pairs, does not necessarily need to be postulated. AID-related structures have been identified in different species of bony fish, including the catfish, based upon their sequence identity to mammalian AID (52, 53). Recent studies have also found that AID from zebrafish and fugu are able to catalyze class switch recombination in mouse B cells. In addition, mutator activity was demonstrated by reversion of an inactive kanamycin allele in Escherichia coli and inactivation of ura3 in Saccharomyces cerevisiae (54). Thus, AID-related structures in bony fish appear to have functional activity.

A central question of somatic hypermutation is whether somatic mutation serves to alter the ability of expressed Ab H and L chains to bind Ag. The related second question is whether a mechanism exists that can preferentially select the B cells with the mutated higher affinity binding sites such that these populations predominant the immune response. In mammals Ag-stimulated, class-switched B cells proliferate and undergo somatic mutation in germinal centers wherein mutated lineages with higher affinity receptors compete for limited amounts of Ag and are selectively expanded while cells with less effective binding sites undergo apoptosis (Refs.20 and 21 , see also Ref.55). The channel catfish, as well as other bony fish, does not undergo class switching, and neither lymph nodes nor germinal centers are present. Because affinity maturation may occur in mammals in the absence of germinal centers (22, 23, 24, 25, 26, 27), it was important to determine whether selection mechanisms could be detected by analyzing the patterns of mutation that occurred in catfish H chains. These studies, in contrast to those in mammals, found no supporting evidence to suggest that CDR-targeted replacement mutations result in selection. First, these studies showed that there was no significant difference in the positional distribution of the mutations in VH-encoded codons. Secondly, the general result that CDRT was significantly more mutable than FRT was predominantly attributed to the skewed distribution of the highly mutable codon AGC. Mutations in AGC accounted for >40% of the mutations in CDRT, and distribution analyses of the serine codons AGC, AGT, and TCN showed that the AGC was preferentially located in CDR regions. In this regard, these analyses phylogenetically underscore the studies of Wagner et al. (56) who also concluded that mutations are inherently targeted toward CDR regions because of the nonrandom distribution of the AGC codon. Furthermore, when the R:S ratios of mutations occurring in FRT were compared with those in CDRT there was no significant difference. There was also no difference between FRT and CDRT when the R:S ratios of the mutations found only in the AGC codon were compared. The higher ratio of R:S substitutions in CDR as compared with FR has been used as an indication for Ag selection (45); thus, by these criteria, mechanisms targeted toward selection of replacement mutations in CDR do not appear to be present.

The multinomial distribution method was also used to evaluate the question of B cell selection by Ag, and this model addresses two important features. The first is whether selection mechanisms serve to conserve the basic framework structure of the H chain by selecting for synonymous substitutions. The second is whether selection mechanisms serve to select for nonsynonymous substitutions within the CDR regions that may alter Ag binding. In our analyses, only 10 of the 93 productive rearrangements that had mutations showed evidence for significant scarcity of FR replacements. Of the 51 rearrangements that had four or more mutations in the FR1 to FR3 regions, only seven of these had significant scarcity of FR replacements (13.7%), and four of these were within the VH-encoded region of clonal set VH7B-CS2. In comparison, 66% of the H chains in B cell lymphomas (57), 36–84% of the H chains in tumor-infiltrating B cells (58), and 72–82% of the H chains in synovial B cells (59) were significant for scarcity of FR replacements and/or excess of CDR replacements.

The analyses of the VH7B-CS2 clonal set proved especially informative. This set, composed of seven clones, exhibited extensive mutations when these clones were compared with the VH clonal set progenitor sequence (Fig. 2C). In this set, clone 1E10 was the immediate precursor to three other clones (3B08, 6F08, and 6H01). 1E10 was significant for scarcity of FR replacements in the VH-encoded region as were two of its descendants (6F08 and 3B08). The third descendant 6H01, which had acquired two additional mutations, was no longer significant for scarcity of VH FR replacements. Similarly, two descendants (6D07 and 6E07) that arose from branches different from those leading to 1E10 had accumulated a total 17 or 10 mutations, respectively, from the progenitor sequence; but neither of these exhibited significant scarcity of FR replacements. Lastly, when the multinomial distribution studies were expanded to include the CDR3 and FR4 regions, only 1 clone in this clonal set (3B08) showed evidence for significant scarcity of FR replacements. Thus, if a significant positive selective force for synonymous FR substitutions exists, it is not uniformly evident even within members of the same clonal set.

Only 1 of the 93 productive rearrangements exhibited significant excess of nonsynonymous CDR substitutions. This single clone (2D12AVH10) had four total VH mutations with two of the three mutations within the CDR resulting in replacements. This minimal result strongly indicates that positive selection mechanisms that serve to enrich B cells based upon nonsynonymous substitutions in CDR do not appear to be functional in bony fish. This conclusion is in agreement with other studies in bony fish that have shown that the affinity of serum Ab population varies but does not significantly increase with time postimmunization when compared with the 3- to 4-log increase in affinity typically observed in mammals (60, 61, 62, 63, 64). In channel catfish, the affinity of the serum anti-DNP Ab population was measured by equilibrium dialysis in samples from individual animals over a 2-year period. These results showed that during this extended time period, there was less than a 1-log increase in the affinity of the Ab population (62). These studies also detected low-affinity sites in the Ab population at each time point, and Sips analysis of Ab heterogeneity did not show a significant decrease during the immunization period. These results appear to be consistent with the present analyses on the divergence patterns of members in clonal sets. Mutations appear to accumulate during clonal expansion, and sublineages that presumably diverged early in B cell clonal expansion and do not exhibit significant levels of CDR-targeted replacements remain present in the B cell population. Thus, these results indicate that somatic mutation may have evolved as a mechanism to principally increase repertoire diversity. This basic mechanism continues to be phylogenetically operational as shown, for example, in the repertoire studies with sheep (65) and the apparent lack of selection in the studies with Xenopus H chains (28).

In conclusion, somatic mutation occurs within catfish H chain V regions. The analysis of these hotspot motifs has shown that although these targets share common motifs, their number is restricted when compared with the spectrum of mutational targets known in mammals. It will be of interest to determine whether these differences are due to variant enzymatic activities of the AID-related molecules in bony fish or whether these differences are attributable to AID-associated factors that may chaperone AID to hotspot motifs, such as have been suggested in the studies with replication protein A (66). Lastly, these studies found no substantial evidence to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the Ig and Ab repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.


   Acknowledgments
 
We extend appreciation to Drs. Edward F. Meydrech and Jacob Olivier for statistical consultation, Mary Duke for high throughput cDNA sequencing support, and Miles Lange for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the National Institutes of Health (AI23052). Back

2 Address correspondence and reprint requests to Dr. Craig J. Lobb, University of Mississippi Medical Center, Department of Microbiology, 2500 North State Street, Jackson, MS 39216-4505. E-mail address: clobb{at}microbio.umsmed.edu Back

3 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; CDRT, the total nucleotides or codons encoded within CDR1 and CDR2; DH, heavy chain diversity region gene segment; FR, framework region; FRT, the total nucleotides or codons encoded within FR1, FR2, and FR3; JH, heavy chain joining region gene segment; R, replacement (nonsynonymous) substitution; R:S, the ratio of the number of replacement to silent substitutions; S, silent (synonymous) substitution; VH, heavy chain variable region gene segment. Back

Received for publication October 17, 2005. Accepted for publication November 10, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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