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* Department of Molecular Genetic and Cell Biology,
Committee on Immunology, and
Department of Statistics, University of Chicago, Chicago, IL 60637
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Msh2/Ung double-knockout mice produce essentially only C and G transitions during SHM and have severely reduced CSR (23). We initiated crosses between Msh6–/– and Ung–/– mice to test whether MMR and BER are the major mechanisms of creating post-AID mutations from A and T, and transversions from C and G. We used Msh6 rather than Msh2 because the former, together with Msh2 in a MutS
complex, is only involved in recognition of single base-pair mismatches and single base loops, whereas Msh2, in combination with Msh3 (MutS
), is also involved in recognizing larger loops (24). AID creates single base U/G mismatches; larger mismatches and unpaired loops are not direct consequences of AID deamination, although loops may arise during CSR in R-looped switch regions, and potentially during SHM if AID deaminates two or more consecutive Cs or if error-prone repair causes a mismatch of more than one nucleotide. A potentially different role of the mismatch repair complex MutS (Msh2/Msh3) compared with MutS (Msh2/Msh6) was suggested, because the pattern of switch joints appeared to differ in Msh2–/– and Msh6–/– mice (12). Recently, it was suggested that Msh6 may serve as a scaffold in SHM (25).
The Msh6–/–Ung–/– mice were also important to further study whether the sparing of the very 5' end and the C region of Ig genes during SHM was indeed due to lack of AID access/activity in these regions. We found previously that the lack of mutations in the very 5' and 3' regions of Ig genes was as severe in Ung–/– mice as in wild-type mice and concluded that AID did not function in these regions (26). There remained the possibility that AID-created uracils were present in the 5' and 3' regions of Ung–/– mice, but that U/Gs in these flanking regions were repaired error-free by MMR that proceeded without mistakes, as MMR would elsewhere in the genome. In that case, uracils in the 5' and 3' ends could have been faithfully repaired to cytosines by MMR. Elimination of both single-mismatch MMR and Ung likely reveals whether error-free copying of uracils occurs in Ig gene SHM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Ung–/– mice were a gift of D. Barnes and T. Lindahl (Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, U.K.), and were further bred in our mouse facility on a C57BL/6 background. A male Msh6+/– mouse was obtained from the Mouse Repository at the National Cancer Institute (Rockville, MD); the latter mice were originally donated by W. Edelmann (Albert Einstein College of Medicine, Bronx, NY). Both Ung–/– and Msh6+/– mice had a C57BL/6 x 129 background. The mice were maintained according to National Institutes of Health instructions. (Ung+/–Msh6+/–)F1 mice were acquired by crossing Ung–/– mice with the Msh6+/– mouse. Offspring were further crossed twice to generate Ung–/–Msh6+/+, Ung+/–Msh6–/–, and Ung–/–Msh6–/– mice. The experiments with mice have been reviewed and approved by the University of Chicago Institutional Animal Care and Use Committee.
Cells and DNAs
Mice (all mice were 2 mo old, except for the Ung+/–Msh6–/– mice which were 3 mo old; see Table I) were immunized i.p. with 108 sheep RBC (SRBC; MP Biomedicals) on day 1, boosted with the same amount of SRBC on day 8, and sacrificed on day 11. Sera were saved for ELISA analysis, while spleens were removed and minced in RPMI 1640. RBC in the spleen were lysed and the remaining spleen cells were stained with fluorescein-peanut lectin agglutinin (PNA) and PE-anti-B220. PNAhighB220+ cells were purified using a Mo-Flo sorter (BD Biosciences) at the Immunology Core Facility (University of Chicago). The purified cells were lysed in lysis buffer containing proteinase K (500 µg/ml), EDTA (50 mM, pH 8.0), SDS (1%), and Tris (50 mM, pH8.0) overnight at 37°C. DNAs were extracted using phenol/chloroform.
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The PCR method for the H chain gene was described elsewhere (26, 27). Briefly, PFU turbo DNA polymerase (Invitrogen Life Technologies) was used during a touchdown PCR in the presence of a pair of primers which specifically annealed to the rearranged VJH558 region and the JH4-Cµ intron (27). Gel electrophoresis was performed to purify a 1.2-kb DNA fragment containing VJH558 rearranged to JH4 using the QIAquick Gel Extraction kit (Qiagen). The purified DNA was inserted into a TOPO II (blunt) vector (Invitrogen Life Technologies). The vector-transformed bacteria were grown overnight at 37°C in a kanamycin plate. Colonies were picked and further amplified in Luria-Bertani medium in the presence of kanamycin. The vector DNA was purified using the QIAprep Spin Miniprep kit (Qiagen). Alternatively, colonies were picked and DNA was automatically prepared at the sequencing center (University of Chicago). DNA was sequenced using automated sequencing (3730XL; Applied Biosystems), and sequence analysis was performed using Sequencher 4.1. Only mutations in the 1.1-kb DNA sequence starting at the first nucleotide of the intron (
0.7 kb from the promoter) were counted to avoid any ambiguity caused by VDJ recombination. For the sequencing of the 5' end of the 
1 gene, the procedure described in Ref. 26 was used.
ELISA
ELISA was performed using the SBA clonotyping System/AP kit according to the manufacturer’s instructions (Southern Biotechnology Associates). Briefly, 5 µg of capture Ab was added to each well in an ELISA plate, and the plate was incubated overnight at 4°C. After rinsing the plate three times with PBS containing 0.05% Tween 20 to remove the extra capture Ab, the wells were blocked with 1% BSA. The diluted serum samples containing Igs of interest were then added to the wells, and the ELISA plate was incubated overnight at 4°C. Unbound serum proteins were rinsed out with PBS containing 0.05% Tween 20 before various Abs anti-Ig C regions conjugated with alkaline phosphatase were added. The plate was incubated at room temperature on a shaker for 1 h. Unbound Abs were removed by rinsing the plate five times with PBS containing 0.05% Tween 20. The substrate (p-nitrophenyl phosphate) was added to the wells and the OD was read at 405 nm in the Synergy HT reader (Bio-Tek Instruments) at various time periods. Each serum sample was assayed three times in duplicate. Fig. 2 shows the average of all assays.
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Pairwise comparisons of Ig levels in different transgenic mice were performed using a rank-based nonparametric testing procedure. For each experiment, the Ig levels of the mice were ranked, and the sum of ranks for one of the strains was calculated across experiments. An empirical distribution of this statistic under the null hypothesis of no difference in Ig levels was constructed using simulations, and two-sided p values were calculated from this distribution. We performed 10,000 simulations, and each simulated rank was drawn by assuming that the Ig levels of the two strains of mice are independent identically distributed variables within each experiment. The statistically significant differences are indicated in Fig. 2.
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Results |
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We analyzed mutations in the H chain locus starting from the first nucleotide of the JC intron of VDJH4-rearranged genes. The uracil-DNA glycosylase-deficient mice underwent efficient somatic hypermutation indicated by the mutation frequencies in mutated DNA sequences (3.3 and 2.3/1000 bp, compared with 2.6 and 2.5/1000 bp in wild-type mice) (Table I). However, the overall mutation frequencies in mice with the Msh6–/– background and wild-type Ung were reduced (1.4, 1.5, 1.3, and 1.5/1000 bp in mutated DNA sequences) (Table I) and mutated sequences accumulated only few mutations (Fig. 1, pie charts). The unchanged mutation frequencies in Ung–/– mice and the reduced mutations in the secondary response of Msh6–/– mice agrees with previous findings in Ung–/– mice (5, 26), Msh6–/– mice (7, 9), and Msh2–/– mice (10), respectively. Thus, production of Msh6, as well as Msh2, which together are involved in mismatch repair of single base mismatches or loops, appears to increase the mutation frequency. Alternatively, Msh2/Msh6 may aid the survival of mutating B cells (10) (see Discussion). Interestingly, in Msh6–/–Ung–/– double-knockout mice the mutation frequencies in mutated sequences were as in wild-type mice (2.7, 2.1, 2.3, and 2.9/1000 bp of mutated sequences) (Table I) and many mutated sequences accumulated large numbers of mutations (Fig. 1, pie charts) (see Discussion).
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In Ung–/– mice, as described before (5, 26), most of the mutations from C and G were transitions. The Ung–/– and wild-type mice showed a similar ratio of mutations at A over those at T, however, the A,T mutation frequency was reduced in the Ung–/– mice (Fig. 1). The percentage of A,T mutations in the 1100 nt 3' of JH4 was 34%, compared with 51% in the wild-type mice (Table II, no. 6), a reduction in Ung–/– by 33%. This is a similar reduction of A,T mutations as previously seen near JH4 in the same Ung–/– mice (5, 32) which had 43% A,T mutations, compared with 57% in wild-type mice (a reduction by 25%) (Table II, no. 4). Our findings here are also similar to the A,T mutations in the 1145 nt 3' of JH4 in another study (26). There, wild-type mice had 47% A,T mutations, and Ung–/– mice only 36% (Table II, no. 3). The least reduction of A,T mutations by only 11% was seen in the most highly mutated region of a 
transgene (Table II, no. 1) (26). The highest reduction of A,T mutations (by 52%) was found toward the 3' end of the JC intron of the same transgene (Table II, no. 7) (26). Because in this study (26), all regions were sequenced from PNAhigh B cells of the same mice (Table II, nos. 1, 2, 3, 5, 7), but the proportion of A,T mutations in Ung–/– vs wild-type mice vary from 0.48 to 0.89, the differences are unlikely due to immunization, but rather may depend on the primary sequence. Taken together, these findings suggest that Ung, and thus presumably BER, plays a role in A,T mutations (see Discussion).
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A previous study showed that C,G mutations were mainly transitions and A,T mutations were greatly diminished in Ung–/–Msh2–/– double-knockout mice (23). To further determine whether the Msh2/6 complex is essential and whether there is any contribution by the Msh2/Msh3 complex, or Msh2 alone, to generate transversions from C,G or mutations from A,T, we analyzed Ung–/–Msh6–/– mice along with Ung+/–Msh6–/– mice as controls (Fig. 1). Only 2% of the mutations in the Ung–/–Msh6–/– mice were at A or T, the rest were C,G mutations, all of them being C>T and G>A transitions. Thus, the findings in the Ung–/–Msh6–/– are very similar to the mutation profile in the Ung–/–Msh2–/– mice, indicating that the Msh2/6 complex, but not the Msh2/Msh3 complex, is required for generating high levels of A,T mutations. A less severe reduction of A,T mutations was seen in Msh6–/– mice with wild-type Ung supporting the notion that some mutations at A and T can arise by Ung-dependent BER without the help of MMR (but see Discussion) (Fig. 1).
CSR is diminished in Ung–/–Msh6–/– mice
To ascertain whether CSR was impaired in the Ung–/–Msh6–/– mice, we conducted ELISA analyses of the sera of SRBC-immunized mice. Data in Fig. 2 are based on the pool of two sets of ELISA data from two groups of mice (each group has a wild-type, an Ung–/–Msh6+/+, an Ung+/–Msh6–/–, and an Ung–/–Msh6–/– mouse). Compared with the wild-type mice, Ung–/–Msh6+/+ mice had significantly lower concentrations of switched Igs (IgG1, IgG2a, IgG3 and IgA; p < 0.0002), except for IgG2b (p < 0.1376), indicating that CSR in the Ung–/–Msh6+/+ mice was significantly impaired (Fig. 2). In Ung–/–Msh6–/– mice serum IgG and IgA were essentially absent. Compared with wild-type mice, in the Ung–/–Msh6–/– double-knockout mice the IgG1, 2a, 2b, 3, and IgA levels were 12–120 times lower (p < 0.0002) (Fig. 2), indicating that CSR is greatly diminished. CSR in the Ung+/–Msh6–/– mice, however, is not severely impaired, except for IgA (p < 0.0002). This differs from previous data that showed that both IgG1 and IgG3 in Msh6–/– mouse sera were significantly reduced (7). The mice in that study were 3–6 mo old, our Msh6–/–Ung+/– mice were 3 mo old (Table I). Because at these ages residual maternal Abs are unlikely, perhaps unknown background genes are involved in the different findings. The higher IgM titers in Ung–/–Msh6+/+, Ung+/–Msh6–/–, and Ung–/–Msh6–/– mice were expected (Fig. 2) to compensate for the reduced or absent IgGs and IgA by overexpressing IgM. Interestingly, although CSR in the Ung–/–Msh6+/+ mice was significantly reduced compared with wild-type mice, it was higher than in Ung–/–Msh6–/– mice (p < 0.0002) (Fig. 2), indicating that both BER and MMR are responsible for CSR and that their roles in CSR may be somewhat overlapping.
The 5' region of Ig genes is not accessible to AID
In a previous study, we showed that, as in wild-type mice, mutations in Ung–/– mice start 100–200 bp downstream of the Ig promoter (26). This finding suggested that AID did not gain access to the very 5' end of Ig genes, because, if it did, in the absence of Ung one would have expected an AID "footprint" of C:G to T:A transitions (26). However, the possibility remained that C,G transition mutations were not seen because mismatch repair recognized potential AID-created uracils and resulted in faithful, error-free restitutions of cytosines. To address this question, we analyzed mutations in the 5' end of Ig genes in the Ung–/–Msh6–/– mice. Of 57 mutations in the Ig L chain gene from 61 clones, no mutations occurred in the first 97 bp and full mutation activity started only 200 bp downstream of the promoter (Fig. 3). This distribution of mutations is the same as in wild-type or Ung–/– mice (26). Because BER and MMR, the major apparent mechanisms responsible for posturacil SHM events, were absent in these mice, we conclude that AID, indeed, is unable to access the 5' region of Ig genes in vivo.
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Mutations are also absent in the 3' end of Ig genes, both in wild-type and Ung–/– mice, suggesting that AID does not act beyond 1.5–2 kb from the promoter (26). To assess the possibility that high fidelity mismatch repair may eliminate uracils in this region, we compared the mutation distributions in the 1.1-kb sequence 3' of JH4. In both the Ung–/–Msh6–/– and wild-type mice, the highest density of mutations was at the 5' end of the JC intron; more 3' the density of mutations was gradually reduced so that mutations at the 3' end of the 1.1-kb region 3' of JH4 were scarce, both in Msh6–/–Ung–/– and wild-type mice (Fig. 4, C and D). Two factors might contribute to the mutation distribution in this 1.1-kb DNA sequence: the distribution of cytosines and the occurrence of the WRC/GYW hotspots. Comparing the 5' end to the 3' end of the 1.1-kb DNA fragment, the frequency of cytosines is similar (Fig. 4A), while the frequency of WRC/GYW hotspots is actually slightly higher 3' (Fig. 4B), indicating that the distribution of mutations is independent of either the distribution of cytosines or the occurrence of AID hotspots. Thus, increasing scarcity of AID-induced deaminations appears responsible for the waning mutagenesis toward the 3' end, suggesting that, indeed, AID does not access or does not act at the 3' end of Ig genes.
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Discussion |
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complex, Msh2/Msh6, that is responsible for recognizing single-base mismatches and loops, and not the MutS- complex, Msh2/Msh3, that recognizes larger mismatches and loops. The fact that almost all mutations at C and G are transitions suggests that they arise by direct replication of the AID created uracils and that, besides BER and MMR, no other repair mechanism may recognize and process the uracils. It had been shown, by us and others, that nucleotide excision repair deficiency does not alter SHM (28, 29, 30, 31). The findings here confirm this. Some mutations at A and T still arise in the double-knockout mice, 2% in the current study (Fig. 1). This is unlikely due to MutS- (Msh2/Msh3) activity, because a similar proportion is seen in Msh2/Ung double-knockout mice (23). Thus, perhaps other uracil glycosylases can get involved (32) and, in rare occasions, their action during SHM may attract error-prone polymerases.
The mutation frequencies in mutated DNA clones are the same in Msh6–/–Ung–/– mice as in wild-type mice (Table I). In contrast, in Msh6–/– mice with wild-type Ung, the mutation frequencies are reduced (bold in Table I). Reduction in mutation frequencies has also been observed in mice with Msh2 deficiency (10, 11) but not in mice with Msh2/Ung double deficiency (23). Rada et al. (23) suggested that, in the presence of Ung, CSR was initiated but when MutS- was absent, CSR was not completed and thus the cells may have died, while in the absence of Ung, CSR would not be initiated. There may also be an interesting alternative explanation, because CSR is not severely compromised in Msh6–/– mice. Perhaps, in the presence of Ung but absence of mismatch repair, many of the uracils created by AID may be repaired to cytosine by the normal base excision repair with polymerase , thus reducing mutations.
CSR is greatly curtailed in the double-knockout mice, however, it is not eliminated (Fig. 2). There clearly are switched Igs for IgG1, IgG2b, and IgA. Perhaps IgG2a and IgG3 were also present at levels not detectable, because these Ig isotypes are also rare in wild-type mice. Presumably, switch recombination in these cases results from spontaneous single-strand breaks near each other, one on each DNA strand, in Sµ and a downstream S region. If this were the case, it would suggest that in normal CSR the recruitment of nonhomologous end joining complexes and the synapsing of Sµ and another S region would not require participation of AID. In fact, in the absence of AID some IgG1 and IgG2a was observed in AID–/– mice (33).
Ung–/– mice had been shown before to retain a high frequency of mutations at A and T (5, 26). This was interpreted to indicate that Ung and consequently base excision repair are not responsible for A,T mutations. Inspecting A,T mutations in the different studies, however, shows that mutations at A and T are consistently lower in Ung–/– mice than in wild-type mice, being reduced by 11–52% of the frequencies in wild-type littermates (Table II). This suggests that base excision repair likely does play a role in A,T mutations. Based on the data in Table II, potentially, up to 52% of A,T mutations arise by an error-prone long patch excision during BER initiated by Ung. Outside of SHM, such long patch BER is relatively error-free due to copying of the G-containing strand by pol or a replicative polymerase, 
or 
(24). In SHM, error-prone polymerases apparently become involved. The proportions of A,T mutations in Ung–/– vs wild-type mice vary greatly in the different DNA sequences analyzed (Table II). This seems unrelated to the proportion of AID hotspots as would be expected if it were due to factors acting after AID. The increasing reductions of A,T mutations are slightly parallel with increasing proportions of known hotspots of the error-prone polymerase 
(WA, and its inverse TW) (34). This may suggest that pol is not the major polymerase interacting with MMR in SHM in the absence of Ung, although it has been reported that pol can interact with Msh2/Msh6 (35).
In the absence of Msh6 but the presence of wild-type Ung, there are very few mutations from A and T, although more than when both Ung and Msh6 are inactivated (Fig. 1; (7, 9). This scarcity of A,T mutations in mice deficient for Msh6 alone disagrees with the conclusion that Ung is responsible for a proportion of up to 52% of mutations from A and T. However, this finding hints at a collaboration between Ung and MMR (26). Ung is more active on ssDNA than dsDNA (36, 37). Presumably, in normal SHM, Ung acts efficiently on ssDNA created by Msh2/Msh6 induced excision of either the U or G containing strand. However, because Ung is less active in removing the U in dsDNA, long patch base excision repair is reduced in Msh6–/– mice.
SHM is restricted to transcribed Ig genes between +100 and 200 from the start of transcription to 2 kb 3' (26, 38, 39, 40, 41, 42, 43). This distribution is unaltered in Ung–/– mice (26), suggesting that AID did not access or not operate in the spared 5' and 3' ends of Ig genes. It was, however, possible that AID-induced uracils were produced in the 5' and 3' ends, but that the U/G mismatches were recognized by Msh2/Msh6 and repaired without error. The present study shows that in Msh6–/–, Ung–/– mice the 5' and 3' regions again have few or no mutations (Figs. 3 and 4). It is thus very unlikely that AID targets these regions. This is strikingly different from the result of the cell-free study that showed that AID could access cytidines immediately downstream of the promoter in naked DNAs (44). Therefore, the interesting questions that remain are whether in vivo AID may be associated with transcription complexes only in the +100 nt to +2 kb region of Ig genes, whether it can only access this region even without association with the transcription complex, or whether it is present in complexes with DNA in the 5' and 3' regions but cannot act. These questions are under further study.
AID activity in vitro is much higher than the mutation frequencies observed in SHM in vivo. This may be due partially to lower concentrations of AID in vivo. However, SHM is tested several days after AID induction in vivo, whereas in vitro, very high mutation frequencies are seen within a few minutes (e.g., Ref. 45). Because BER and MMR appear to be the major mechanisms involved in the processing of AID induced uracils in vivo, the transition mutations from C and G in the absence of BER and MMR likely are a "footprint" of AID, namely represent the uracils created by AID. Thus, it appears that AID action might be curtailed in vivo. An untested alternative is that another uracil glycosylase (such as Smug (32)) is involved in removing a high proportion of AID-created uracils during SHM in vivo and that this glycosylase prevents or does not foster the access of error-prone polymerases.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI47380 and AI053130. 
2 Address correspondence and reprint requests to Dr. Ursula Storb, Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637. E-mail address: stor{at}midway.uchicago.edu
3 Abbreviations used in this paper: AID, activation-induced cytosine deaminase; SHM, somatic hypermutation; CSR, class switch recombination; BER, base excision repair; MMR, mismatch repair; SRBC, sheep RBC; PNA, peanut lectin agglutinin.
Received for publication May 23, 2006. Accepted for publication July 20, 2006.
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References |
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1 transgene in young but not older mice. Mol. Immunol. 36: 83-91. [Medline] error spectrum. Nat. Immunol. 2: 530-536. [Medline], suggesting a role for A:T mutations in antibody genes. J. Exp. Med. 201: 6337-6645. 1 kb from V(D)J gene. J. Exp. Med. 172: 1717-1727. This article has been cited by other articles:
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  H. M. Shen, M. G. Poirier, M. J. Allen, J. North, R. Lal, J. Widom, and U. Storb The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription J. Exp. Med., May 11, 2009; 206(5): 1057 - 1071. [Abstract] [Full Text] [PDF]
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 C. Canugovi, M. Samaranayake, and A. S. Bhagwat Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase FASEB J, January 1, 2009; 23(1): 34 - 44. [Abstract] [Full Text] [PDF]
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 P. Pham, K. Zhang, and M. F. Goodman Hypermutation at A/T Sites during G{middle dot}U Mismatch Repair in Vitro by Human B-cell Lysates J. Biol. Chem., November 14, 2008; 283(46): 31754 - 31762. [Abstract] [Full Text] [PDF]
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S. Peron, A. Metin, P. Gardes, M.-A. Alyanakian, E. Sheridan, C. P. Kratz, A. Fischer, and A. Durandy Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination J. Exp. Med., October 27, 2008; 205(11): 2465 - 2472. [Abstract] [Full Text] [PDF]
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J. M. Di Noia, G. T. Williams, D. T.Y. Chan, J.-M. Buerstedde, G. S. Baldwin, and M. S. Neuberger Dependence of antibody gene diversification on uracil excision J. Exp. Med., December 24, 2007; 204(13): 3209 - 3219. [Abstract] [Full Text] [PDF]
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P. Langerak, A. O.H. Nygren, P. H.L. Krijger, P. C.M. van den Berk, and H. Jacobs A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification J. Exp. Med., August 6, 2007; 204(8): 1989 - 1998. [Abstract] [Full Text] [PDF]
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 L. Ohm-Laursen and T. Barington Analysis of 6912 Unselected Somatic Hypermutations in Human VDJ Rearrangements Reveals Lack of Strand Specificity and Correlation between Phase II Substitution Rates and Distance to the Nearest 3' Activation-Induced Cytidine Deaminase Target J. Immunol., April 1, 2007; 178(7): 4322 - 4334. [Abstract] [Full Text] [PDF]
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M. S. Neuberger and C. Rada Somatic hypermutation: activation-induced deaminase for C/G followed by polymerase {eta} for A/T J. Exp. Med., January 22, 2007; 204(1): 7 - 10. [Abstract] [Full Text] [PDF]
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, Wild type; 
, Ung–/–Msh6+/+; 
, Ung+/–Msh6–/–; 
, Ung–/–Msh6–/–. The statistical significance is indicated directly above a column (when less than wild-type mice) or higher up above a columns (when more than wild-type mice). *, Value of p 
0.0002 compared with wild-type. #, Value of p 
