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in Somatic Hypermutation Determined by Analysis of Mutations in a Patient with Xeroderma Pigmentosum Variant
* Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892; and
Basic Research Laboratory, National Cancer Institute, Bethesda, MD 20892
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Abstract |
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(pol )
in somatic hypermutation of B cells, a mutational analysis of 24
nonproductive rearrangements from a patient with xeroderma pigmentosum
variant with a defect in pol was conducted. Although the mutational
frequency of A and T bases decreased in WA (A/T, A) motifs, regardless
of their RGYW (purine, G; pyrimidine, A/T) context, the overall
mutational frequency of A or T bases was not affected. Moreover, the
overall mutational frequency of the sequences examined was not
decreased. There was an apparent increase in the number of insertions
and deletions. The results are consistent with the conclusion that pol
specifically targets WA motifs. However, its overall contribution
to the somatic hypermutational process does not appear to be
indispensable and in its absence other mechanisms maintain mutational
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it has been shown that variable but not constant regions of Ig genes contain a high frequency of double strand breaks near RGYW hotspots, suggesting that error-prone DNA polymerases might be involved in creating mutations during the repair of these DNA breaks (7, 8, 9). To date, many error-prone DNA polymerases have been described, and based on their expression and biochemical properties, a number could be involved in somatic hypermutation of Ig genes individually or in combination.
Xeroderma pigmentosum (XP) is a rare inherited disease with a 1000-fold
increase in the incidence of UV-induced skin cancer in association with
defective DNA repair (10, 11). Many XP patients have
defective nucleotide excision repair, whereas XP variant (XPV) patients
have normal nucleotide excision repair but are defective in their
replication of UV-damaged DNA because of a defect in DNA polymerase
(pol ) (12, 13). Pol can efficiently bypass a
thymine-thymine cis-syn cyclobutone T-T dimer in the
presence of dATP (14, 15, 16, 17). In XPV, UV-induced nucleotide
damage is repaired by nucleotide excision repair or by error-prone DNA
polymerase 
(pol ). Even though pol reads through T-T dimers
without introducing errors, pol is inaccurate when copying
undamaged DNA and incorporates errors at a frequency of
10-2 (17).
Recent reports have implied that pol might be involved in somatic
hypermutation of Ig genes. Zeng et al. (18) reported that
somatic hypermutation in patients with XPV occurs at the normal
frequency but in an altered pattern with decreased mutations of A and
T. In addition, Rogozin et al. (19) showed that pol
introduces mutation in WA (AT/A) motifs (with the
underlined nucleotide being most frequently mutated). These results
supported the idea that pol might play a role in somatic
hypermutation. However, many WA mutations were part of RGYW
motifs and therefore could have been targeted by the germinal center
RGYW-focused mutator (20). Therefore, it remained
uncertain whether some apparent WA mutations were actually
RGYW mutations. Indeed, the impact of pol deficiency on
WA mutation specifically was not analyzed. Furthermore,
only mutations in productive rearrangements of XPV patients
were analyzed. As a result, it was difficult to be certain that
differences in the pattern of mutated nucleotides reflected the action
of the mutational machinery or rather the influences of subsequent
selection.
To address these issues, we analyzed the mutational characteristics of
nonproductively rearranged Ig H chain variable region
(VH) genes isolated from individual peripheral B
cells of a patient with XPV. Mutations in nonproductive
VH rearrangements represent the immediate impact
of the mutational machinery in the absence of selective influences and
should provide a direct indication of the impact of the absence of pol
on mutational frequency and pattern (2).
Results of this study indicate that in the absence of pol , WA
mutations are decreased both in RGYW/WRCY motifs and non-RGYW
sequences. However, the overall frequency of T/A mutations was not
decreased. Even though WA mutations were decreased in XPV, pol does
not seem to have an indispensable impact on mutational activity of Ig
genes because the overall mutational frequency was normal. An apparent
increase in insertions and deletions was found in the nonproductive XPV
repertoire, suggesting that in the absence of pol another
error-prone polymerase that preferentially introduces insertions and
deletions may be operative.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ)
gradient centrifugation from XP31BE, a 55-year-old male patient with
XPV. The DNA repair defect in the patient was classified as XPV based
on clinical features that included pigment changes and multiple skin
cancers in association with normal post-UV survival, normal unscheduled
DNA synthesis, and a mutation in pol .
FACS analysis
Immunofluorescence staining for cell sorting was performed by incubating PBMC with PE-conjugated anti-CD27 (BD PharMingen, San Diego, CA), PerCP-conjugated anti-CD19 (BD Biosciences, San Jose, CA), and FITC-conjugated anti-CD3. CD19+CD27+CD3- memory B cells and CD19+ B cells were sorted using a FACSVantage (BD Biosciences).
Amplification and sequencing of rearranged VH genes
Rearranged VH genes of individual CD27+CD19+ memory and CD19+ B cells were specifically amplified from genomic DNA, followed by direct sequencing as described (2, 6). Sequences were analyzed using the V Base Directory to identify the respective germline genes. Of 123 VH genes sequenced, 24 nonproductive rearrangements containing mutations and 52 productively rearranged VH genes with mutations were compared. No clonally related sequences were detected. A total of 94 mutated Ig genes from normal donors (37 nonproductively and 57 productively rearranged VH genes) were used for comparison (2). Sequences are accessible in the GenBank Nucleotide Sequence Database (pending). The nonproductive rearrangements were either out of frame (21 of 24) or had introduced stop codons at the VDJ junctions (3 of 24). Because of the nature of the internal primers used for amplification, mutations in framework region (FR) 1 could not be assessed. Therefore, analysis of this region was omitted. The maximal error rate of the amplification technique was previously calculated to be 10-4 mutations/bp with the same Taq polymerase used for this study (21).
Statistical analysis
Sequences were analyzed with the
2 test to compare the differences in the
frequencies of mutations and the mutations of RGYW motifs between the
productive and nonproductive repertoires. The expected mutational
frequency of the 15 RGYW/WRCY motifs was 23.6% (22). The
actual frequency of RGYW/WRCY mutations was compared with the expected
random frequency using the goodness of fit
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Distribution of mutations
The 24 nonproductively rearranged VH genes, comprising 5989 bp, contained 247 nucleotide substitutions and a mutational frequency of 4.1 x 10-2 (4.6 x 10-2 for CD27+CD19+ memory B cells vs 3.0 x 10-2 for CD19+ B cells). This was comparable with the previously published (2) data obtained from normal donors (357 substitutions in 9498 bp; 3.8 x 10-2, NS). The 52 productively rearranged VH genes contained 352 nucleotide substitutions within a total of 12,948 bp, for a mutational frequency of 2.7 x 10-2 (3.3 x 10-2 for CD27+CD19+ memory B cells vs 2.0 x 10-2 CD19+ B cells). This frequency was somewhat less than that found in normal donors (484 of 14,474; 3.3 x 10-2, p < 0.003). As has previously been noted in normal individuals (2), the overall mutational frequencies in nonproductive and productive rearrangements were significantly different in XPV (p < 0.0001).
The overall distribution of mutations in specific regions of the VH rearrangements were similar in both nonproductive and productive rearrangements, consisting of increased replacement mutations in complementarity-determining regions (CDRs) compared with FRs. The ratio of replacement to silent mutations was significantly greater in CDRs compared with FRs of the productive rearranged VH genes only (p < 0.0001). This was comparable with the findings of nonproductive and productive VH rearrangements of normal donors (2).
Mutations of RGYW/WRCY
Previously, it has been shown that the RGYW/WRCY motif is a target
for increased mutational activity (2, 22, 23). To examine
the possible role of pol in RGYW/WRCY targeting while avoiding the
influence of selection, the presence of all mutations occurring in
these motifs was analyzed in the nonproductive VH
rearrangements. In the nonproductive sequences, 32% (80 of 247 events)
of the mutations occurred within an RGYW/WRCY motif, whereas in the
productively rearranged sequences, 45% (158 of 352 events) of all
mutations occurred in these motifs. The difference in the frequency of
mutations in RGYW/WRCY motifs between productive and nonproductive
rearrangements is significant (p < 0.002). This
difference showed that mutations in RGYW/WRCY motifs are positively
selected in productive rearrangements as was previously shown in the
normal repertoire (23). To address the potential role of
pol in mutations of WA sequences, all mutations that
affected the TA and AA dinucleotides in the nonproductive
repertoire were analyzed. Altogether, 18 of 247 (7.3%) base
substitutions occurred in WA sequences (Table I
). This frequency was
significantly less than that observed in nonproductive rearrangements
of normal donors (93 of 359; 25.9%) (p <
0.001). The frequency of WA mutations in the XPV sequences was
significantly reduced compared with normal in both RGYW/WRCY motifs
(2.8% vs 13.4%, p < 0.001) and non-RGYW/WRCY sequences
(4.5% vs 12.5%, p < 0.001). Theseresults implied
that pol targets WA sequences independent of their location in
RGYW/WRCY motifs. Analysis of the nature of the 11mutations of WA
dinucleotides outside an RGYW/WRCY motif revealed that A was mutated 9
times (replaced by G three times, T four times, and C twice) and T was
mutated twice (replaced by A twice). This was also consistent with the
pol bias for WA compared with WA
(19).
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Within the nonproductive repertoire, G was mutated most frequently
(34% of all mutations), followed by C (29%), A (17%), and T (20%)
(Tables II, III, and IV), whereas in the
nonproductive repertoire of normal donors
G, C, A, and T accounted for 26, 30, 23,
and 21% of mutations, respectively. The frequency of G
mutations was significantly greater in the nonproductive
repertoire of the patient with XPV than in the normal donors
(p < 0.04). Within the productive rearrangements, G
(34%) and C (30%) were mutated significantly more frequently in the
XPV patient than that was noted in normal donors (G, 26%; C, 24%),
whereas A (18%) and T (19%) were less frequently mutated in the XPV
patient than in the normal donors (T, 24%; A, 26%). These results
suggested that the decrease in T/A mutations noted in the productive
rearrangements reflects selective influences and not the immediate
action of the mutator. Finally, no bias of transitions over
transversions was observed (Tables III and IV).
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To determine whether the germline sequence of individual genes might have affected the results, the distribution of mutations in specific genes was analyzed. Three VH3 genes, VH3–15, VH3–23, and VH3–33, were found sufficiently frequently in the nonproductive repertoire (n = 3, 2, 2, respectively) to permit analysis. These VH rearrangements harbored 73 mutations, of which only 2 (3%) were in WA motifs. Of these 73 mutations, however, 18 (25%) were of A, 17 (23%) were of G, 22 (30%) were of C, and 16 (22%) were of T. Therefore, no indication of a decrease in A or T mutations was observed in this subset of VH genes, as was noted with the entire group of VH genes.
Insertions and deletions
Altogether, 7 of 24 nonproductive rearrangements had insertions
and/or deletions, whereas none was found in the productive
rearrangements (Table V). These events were either one or two base
insertions and/or deletions per sequence. Overall, five base insertions
were observed including two of G, two of T, and one of C insertions. In
addition, five base deletions were noted including two of C and three
of T. All events were in members of theVH3
family, including 2 in 3–33, and one in each 3–7, 3–8, 3–49, 3–73,
and 3–23. Most of the events were observed in the vicinity of an
RGYW motif, either within the motif or +1 to +5 or within -2 of the
motif (Fig. 1). In comparison, a total of
9 base deletions was found in 37 nonproductive rearrangements from
normal individuals, whereas none was found in the
productive rearrangements
(2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Based on a correlation between strand
specific hotspots for somatic mutations at WA sequences and
the error specificity of mammalian DNA pol , Rogozin et al.
(19) suggested that pol might contribute to somatic
hypermutation at A-T base pairs. However, most mutations at
WA motifs are included within the highly targeted RGYW
motifs, raising the possibility that many of the WA
mutations were introduced by the same mutational mechanism that
targeted RGYW motifs. If this were correct, a role for pol in
somatic hypermutation would be considerably diminished. In addition,
pol is down-regulated in vitro in response to AgR signaling in
human tonsil B cells and in CL-01 Burkitt lymphoma cells
(24), whereas it is up-regulated in murine germinal
centers (18) and after stimulation of BL2 Burkitt lymphoma
cells with T cells or IgM cross-linking (25). Because of
these disparate findings, the contribution of pol to somatic
hypermutation is still uncertain. To address these issues, a panel of
24 nonproductively rearranged VH genes with
somatic mutations from a patient with XPV was analyzed and compared
with a previously published panel of 37 nonproductively rearranged
VH genes from normal donors (2).
Mutations in the nonproductive VH genes are
particularly important to analyze because they represent the direct
impact of the hypermutational machinery without subsequent influences
of selection (2). The current study shows that in
the absence of pol , the mutational frequency is not affected
but WA mutations are markedly decreased,
regardless of RGYW/WRCY context. However, targeting of mutations to
RGYW/WRCY motifs was still observed in XPV B cells. The finding of
increased numbers of insertions and deletions in the nonproductive
rearrangements of XPV patient also suggests a possible role of pol
in preventing these events or that some other DNA polymerases may play
a greater role, when pol is not available. The results were
obtained from analysis of nonproductive rearrangements of a single
patient and, therefore, individual idiosyncrasies could have
contributed to the results. However, a previous analysis of
productive VH6 rearrangements from three XPV
patients, including the one studied here, showed marked similarities in
the percentage of mutated sequences, the mutational frequency, and the
distribution of mutations (18), making this possibility
unlikely. Rather, the data suggest that a number of error-prone
polymerases, including pol , can contribute to the process of
somatic hypermutation.
Zeng et al. (18) previously reported that in patients with
XPV, including XP31BE, the patient studied here who lacked pol ,
somatic mutation had an altered pattern with decreased mutations of A
and T. In our analysis, we also noted that A-T were mutated less than
G-C in B cells of the XPV patients, but only in the productive
repertoire. However, in the nonproductive repertoire, this
abnormal pattern was not observed. Therefore, despite the significant
decrease in WA mutations, there was no general decrease in
A or T mutations. Presumably, in the absence of pol , other
mechanisms can target A and T for mutations but apparently specifically
target WA poorly. In the previous study, only productively
rearranged VH6 genes were analyzed (18). It is likely that
the abnormal pattern of mutations reflected altered selection and not
the direct impact of the mutational activity. Notably, however, an
analysis of silent mutations in VH6 genes also
showed diminished targeting to A and T (18), raising the
possibility that the absence of pol may diminish the capacity to
mutate A and T in some circumstances. In contrast, G
substitutions were significantly greater in both the nonproductive and
the productive repertoire of Ig genes in the XPV patient, implying that
the polymerases that were active in the absence of pol may have
targeted G preferentially. A similar result was noted by Zeng et al.
(18).
In another study, Rogozin et al. (19) reported the results
of a mutational analysis of 15 sets of Ig genes as well as the analysis
of the nucleotide substitution pattern of human pol . In this work,
they identified that the hotspot motifs RGYW and
WA were targeted for somatic mutations. Notably, mutations
at WA (TA/AA), which is a
strand-specific hotspot motif in Ig genes, supported a role for an
error-prone polymerase that operated as a mutator during synthesis of
the nontranscribed DNA strand of V regions. In the same study, pol
was identified as being able to cause a biased WA
mutational pattern. The data suggested that pol might contribute to
somatic mutations of Ig genes at A-T bases. The current results support
this possibility, given that WA mutations accounted for 25.9% of all
base substitutions of Ig genes, and there was a significant decrease in
the mutation frequency of this motif in the XPV patient compared with
normal individuals (25.9% vs 7.3%, p < 0.001).
This difference was also observed when we analyzed the WA mutations
that occurred outside an RGYW/WRCY motifs and those that occurred
within RGYW/WRCY motifs. This implies that WA mutations within
RGYW/WRCY motifs are introduced by pol and not by the mechanisms
that introduced mutations in other RGYW/WRCY motifs. The WA mutations
observed prominently involved the 3'-A, consistent with the conclusion
that they resulted from the action of pol . However, even though
WA mutations were significantly diminished in the XPV
patient, there was no overall decrease in the mutational frequency or
change in the frequency of T or A mutations. These results indicate
that pol can play a role in somatic hypermutation, but that role
can be substituted by other mechanisms, presumably involving other
error-prone polymerases.
Another interesting finding was the occurrence of insertions and
deletions around RGYW/WRCY motifs in the nonproductive repertoire but
not in the productive repertoire. Recent studies suggested that
double-strand breaks might be introduced by an unknown
endonuclease with an intrinsic preference for RGYW
motifs (7, 8, 9). As a result of the double-strand breaks,
the intronic enhancer-matrix attachment region complex can be
transiently uncoupled from the promoter complex leading to a block in
transcription. These small gaps may be filled in by DNA
polymerases, which would explain the generation of small duplications,
or exonucleases, which would explain the deletions in some mutated
VHDJH (1).
Numerous template-dependent DNA polymerases have been identified in
mammalian cells (26). The lack of pol may have been
partially compensated for by the creation of mutations by other
polymerases that cause shifts and insertions, as we observed both
insertions and deletions in the sequences from the XPV patient. Based
on its preferential expression in germinal center lymphocytes and
TdT-like activity, pol µ, which causes frameshifts, may be a good
candidate to contribute to this aspect of somatic mutation (26, 27), although a recent report indicates that this polymerase is
not required for somatic mutation in mice (28).
In conclusion, pol affects the hypermutational process at
WA hotspots, but its contribution to overall somatic
hypermutation is not essential. The exact spectrum of polymerases
involved in somatic hypermutation of B cells remains to be completely
delineated.
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Footnotes |
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2 Current address: Department of Internal Medicine, Division of Hematology, University of Istanbul, Istanbul, Turkey.
3 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 9N228, Bethesda, MD 20892. E-mail lipskyp{at}mail.nih.gov
4 Abbreviations used in this paper: MMR, mismatch repair; XP, xeroderma pigmentosum; XPV, XP variant; pol , DNA polymerase ; FR, framework region; CDR, complementarity-determining region.
Received for publication March 26, 2002. Accepted for publication August 1, 2002.
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are dispensable for Ig gene hypermutation. J. Immunol. 168:3702.This article has been cited by other articles:
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