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Department of Histopathology, Guy’s, King’s, and St. Thomas’ School of Medicine, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Investigation of hypermutation by sequence analysis can be confounded by the effects of selection on the mutated Ig genes, because selection has been suggested as the cause of strand bias and "hotspots" (7, 8). Selective influences have been avoided in many studies by looking at noncoding flanking sequences (9), passenger transgenes in mice (10), in vitro systems (11, 12, 13), and unused, out-of-frame, alleles of human Ig genes (7, 14). These studies reveal biases intrinsic to the hypermutation process, which may provide clues as to the mechanism of hypermutation. Hotspots and "coldspots" of mutation have been identified, and sequence motifs that target mutation have been proposed (9, 10, 15, 16). Because these biases were identified by analyzing sequences around all mutations, it was not possible to identify whether there were differences between the sequences around mutated GC and AT bases. We have shown previously that there are characteristic sequences around mutated G’s and C’s, presumably motifs that target the mutation process (14). These are 4-mer motifs with G in the second position and C in the third position. They are consistent with the RGYW motif and many of the dinucleotide and trinucleotide preferences (9, 16). The motif for G is the reverse complement of the motif for C, which is additional evidence for a mechanism that acts on both strands of DNA. We were unable to determine the relationship, if any, between the sequence motifs surrounding mutated A’s and T’s, due to the low numbers of mutated T’s available within the data studied.
In this study, we have extended our previous investigations of human out-of-frame IgH genes using a larger data set and computational analysis which includes all mutations and distinguishes between the different nucleotide substitutions at each base. Inherent biases in Ig gene nucleotide composition were compensated for by calculating the normal base composition flanking A, C, G, and T nucleotides and comparing these values with those obtained for the flanking regions of each different nucleotide substitution. In this way, we have determined sequence motifs surrounding mutated A’s and T’s and have shown that the motifs surrounding a particular mutated base can differ depending upon whether the substitutions are transitions or transversions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The mutated Ig genes were aligned
with the appropriate germline IgVH region (22), and a text
file of the alignment was imported into a Microsoft Excel spreadsheet
(Redmond, WA). Computations of the number of each type of nucleotide
substitution (e.g., A-G, T-C, A-C, etc.) and the composition of the
flanking sequences around these substitutions were performed using
macros in Excel (Visual Basic). The nucleotide distribution in germline
Ig genes is not random; for example, it is rare to have a C in position
-1 from a G. To account for this, the composition of the flanking
sequences around the individual bases (A, C, G, and T) was determined
for the germline genes used in this analysis.
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2 analysis to determine
whether any differences from the norm were significant with 95%
confidence limits. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1 illustrates the differences
between the sequence composition flanking mutations from G and C
compared with the control sequence composition. Significant elements
are illustrated by solid bars. These elements are taken to be a
component of a motif that targets mutation. In this way, the targeting
motif around a mutated G (bold underlined) when all mutations are
considered is [A not C/T], G, [C/T not A/G],
[T not C/G] (Fig. 1c). Similarly, the motif around a
mutated C (bold underlined) when all mutations are considered is [A
not C/G], [A/G not C/T], C, [T not G] (Fig. 1g). This confirms our previous results, which showed that
the targeting motifs for C and G are reverse complements of each other
and are illustrated by the graphs of C and G being reversed and shown
underneath the graphs of G and C, respectively (Fig. 1, c
and d, g and h).
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, a and
b). However, it would appear that the main contribution to
the motif around mutations from C comes from the sequence around C
transitions (Fig. 1, e and f), even though the
numbers of C transitions and transversions are similar
(n = 97 and 84, respectively). Targeting motifs around mutations occurring at A and T
Fig. 2 illustrates the differences
between the sequence composition around mutations from A and T compared
with the control sequence composition. In the same way as before,
significant elements (solid bars) are taken to be a component of a
motif that targets mutation. Taking all mutations from A into account,
the motif is [T not C], A (Fig. 2c). This is very different from the motif around a mutated
T (bold underlined), which is T, T, [A not G],
C when all mutations are taken into account (Fig. 2g).
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, c and d, g and h). In
addition, there was a difference between the motifs around transitions
and transversions when they were calculated separately. In the sequence
around A, the motifs deduced for transitions and transversions were
completely different (Fig. 2, a and b), and there
was only one significant point in the sequence around T (A at position
+1) that was common to both transitions and transversions (Fig. 2, e and f). Strand bias
There is no strand bias seen in mutations from G and C, because
there is no significant difference in the total numbers of mutations
from each (203 and 181, respectively). In addition, when the different
types of mutations are paired with their complementary mutations (G-A
with C-T, etc.), the proportions of mutations within each complementary
pair are equivalent, even though different pairs have different
proportions of mutations (Fig. 3). Fig. 4, a and b, show
that there is no significant difference between complementary G/C
transitions and transversions in individual groups of Ig genes.
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shows that when the complementary A/T pairs
are analyzed, the difference between A-C and T-G and between A-T and
T-A is significant (p = 1 x
10-5 and p = 1 x 10-3,
respectively), whereas the difference between A-G and T-C just fails to
reach significance at the 95% level (p =
0.058). Analysis of the complementary A/T transitions for individual
groups of Ig genes shows that the number of A-G mutations is
significantly higher than T-C mutations in only one group (DP73). DP65,
DP71, and VH32 show higher numbers of T-C than A-G. (Fig. 5a). In contrast, the number
of A transversions is significantly higher than T transversions in
DP63, VH32, DP47, and DP73, and the number of A transversions was
consistently higher than T transversions in all groups, even though the
total numbers of mutations in some of the groups were too small to show
any statistical significance (Fig. 5b). The overall effect
is that the difference between the total number of A transversions and
T transversions is highly significant (p =
5 x 10-8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown previously (14) that the sequences that target
mutation toward G and C are reverse complements of each other, and we
suggested that this is evidence that hypermutation of G’s and C’s
acts on both strands of DNA. This possibility was recently confirmed by
a study in which the number of mutations in individual codons was found
to be correlated with the number of mutations in the corresponding
complementary codon (23). In this study, we have undertaken a more
comprehensive analysis of the sequences flanking mutated bases in a
larger panel of out-of-frame Ig genes and have obtained the patterns of
the flanking sequences for all four bases, and for each type of
substitution individually. Our previous motifs surrounding mutated G’s
and C’s have been confirmed as being 4-mers that are reverse
complements of each other, as one would expect if the mechanism of
mutation was targeted and acted on both strands. Further support for a
strand-independent mechanism is provided by the correlation between the
numbers of individual G mutations and the numbers of their
complementary C mutations (Fig. 3).
The motifs surrounding mutated A’s and T’s are very different from the motifs for G’s and C’s and do not bear the same reverse complement relationship. That we are able to show some significant elements of a motif indicates that there is a targeting component in the AT mutator. However, statistical analysis showed that the elements of the motifs around mutated G’s and C’s reached a much higher level of significance than for the sequence around mutated A’s and T’s, indicating that the AT mutator has less need of a target sequence. It should be remembered that there are many mutations, even from G or C nucleotides, which do not fall within targeting motifs. Therefore, it is likely that there are nontargeted mutators whose effects may be superimposed over any targeted mutators when observing the results of hypermutation.
The large difference between the motif for T and the motif for A, and
the fact that the numbers of individual A mutations are consistently
higher than their complementary T mutations (Fig. 3), is consistent
with a mechanism acting on only one strand of DNA. The observation of
strand bias in these out-of-frame genes would confirm that this is an
intrinsic part of the hypermutation process and not an effect of
selection. There is a small element of reverse complement in the
motifs, at position -1 from A compared with position +1 from T ([T
not C], A is the reverse complement of
T, [A not G]), which might mean that some
mutations from A and T occur as the result of a mechanism acting on
both strands. However, when the mutations are divided into transitions
and transversions, there is no similarity between the resulting motif
from one and the reverse complement of the motif from the other (Fig. 2).
The differences between the sequence motifs surrounding mutated G/C’s
and mutated A/T’s and the identification of strand bias in A/T’s only
suggest that the GC mutator is largely a targeted mechanism that acts
on both strands of DNA, whereas the AT mutator acts mainly on one
strand of DNA only and is less dependent upon a target sequence. Other
evidence for independent GC and AT mutators exists; in shark IgM and
Xenopus Ig, mutations are biased toward GC (24), and a cell
line harboring a mutator that preferentially targets GC base pairs has
been reported (11). IgV genes from mice deficient in the Msh2 mismatch
repair protein show a bias toward GC mutations (25), as do Ig genes
from a Burkitt’s lymphoma cell line (13) and Bcl2 genes
translocated to the Ig locus (26). We have also found an example of a
bias in mutation at AT base pairs in an in vitro mouse Ig system (12)
(Table II).
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When the motifs surrounding the different G substitutions were
determined, there was no major difference between the motif surrounding
a G transversion and that around a G transition (Fig. 1, a
and b). However, the same could not be said for the motifs
surrounding C transversions and transitions, where the main
contribution to the targeting motif for C comes from the sequence
around C-T mutations (Fig. 1, e and f). This
contrast implies a difference in the mechanism by which G and C are
mutated, albeit a small one. In this context, it is interesting to note
that although the numbers of G and C transitions are variable and most
likely equal, a consistent (if not significant) increase of G
transversions over C transversions is seen for all groups of Ig genes
studied (Fig. 4).
The motifs around mutated A’s and T’s vary greatly depending upon
whether transversions or transitions are analyzed. Although the
significant elements in the A motif are both in position -1 from the
mutated A, they are very different from one another (Fig. 2, a and b). Similarly, of the four significant
elements of the motif around mutated T’s, only the A in position +1 is
common to both transitions and transversions (Fig. 2, e and
f). Interestingly, when only transitions from A and T are
considered, strand bias is much less apparent. The difference between
A-G and T-C mutations was not consistent between the different groups
of Ig genes and just failed to reach significance on the total number
of mutations (p = 0.058). The difference
between the numbers of A transversions and the numbers of T
transversions, however, is highly significant
(p = 5 x 10-8), and is
consistent between different groups of Ig genes (Fig. 5). This
difference between transitions and transversions may account for a
previous report where strand bias was not detected in out-of-frame
human Ig genes (7).
The fact that the motifs around the mutated bases can be different, depending upon whether transitions or transversions are used for the analysis, coupled with the observation that strand bias is observed more for transversions than transitions, implies that there may be different mechanisms acting within the GC and AT mutators. Whether the mechanisms that cause these differences are ones of mutation and/or of repair is not known.
In conclusion, close study of the patterns of hypermutation that occur in vivo in humans reveal characteristics that suggest that multiple mutators act on Ig genes. Some (but not all) of these characteristics are recapitulated to varying degrees in artificial systems. These systems may have inadvertently separated components of the hypermutation mechanism and therefore will be useful in elucidating the individual mechanisms that contribute to the whole.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Deborah K. Dunn-Walters, Department of Histopathology, Guy’s, King’s, and St. Thomas’ School of Medicine, St. Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, U.K. E-mail address:
Received for publication December 28, 1998. Accepted for publication March 10, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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