Abnormalities of regulatory genes in early B-cell development often lead to lymphomagenesis. Our previous study showed that there is an abnormal transient expansion of bone marrow (BM) pre-B cells in lymphoma-prone SL/Kh strain mice. Such expansion is a genetic property of SL/Kh stem cells rather than BM microenvironments. Using the percentage of BP1+B220+ pre-B cells in total BM lymphoid cells as a quantitative parameter, we studied the genetic control of BM pre-B cells in 159 F2 offspring of crosses between SL/Kh and NFS/N mice and 334 back-crosses to SL/Kh mice. A highly significant quantitative trait locus was identified on the distal segment of chromosome 3, showing logarithm of odds scores of 22.7 in the F2 cohort and 10.7 in back-cross mice. This quantitative trait locus, named bone marrow pre-B-1, colocalized with lymphoid enhancer factor-1, which encodes a high mobility group DNA-binding protein that is expressed in T and pre-B cells.

SL/Kh is an inbred strain of mouse with a high incidence of pre-B lymphomas spontaneously developing at 3–6 months of age (1, 2). SL/Kh mice exhibit an abnormal transient expansion of BM3 pre-B cells (3), reaching a peak at 4–6 weeks of age. Such expansion is normally not found in other strains of mice but is observed in mice with conditions predisposing them to B lymphomagenesis, such as Eμ-myc transgenic mice (4) and Abelson virus-injected mice (5). Although the etiological agent for SL/Kh pre-B lymphomas is endogenous murine leukemia virus, inhibition of virus expression, either by crossing with Fv4R congenic mice or by injection of maternal resistance factor, does not affect pre-B-cell expansion (3). Lethally irradiated mice restored by SL/Kh BM cells but not by others showed pre-B-cell expansion (3). The SL/Kh phenotype, therefore, seems to be a genetic property of BM stem cells. Here, we attempted to identify and map the SL/Kh host gene that is responsible for pre-B-cell expansion in prelymphomatous SL/Kh BM.

Mice.

The origins and genetic backgrounds of SL/Kh and NFS/N strain mice were described previously (6, 7). F1 and F2 mice and back-crosses to SL/Kh were obtained by appropriate matings. All mice were used at 4 weeks of age unless otherwise noted.

Flow Cytometry.

The single-cell suspension was prepared from the right femur BM plug and adjusted to 106 cells/ml. Total BM cell yield was in the range of 0.8 × 107-1.2 × 107 cells per femur, and no significant difference was noted between SL/Kh and NFS/N mice at 4 weeks of age. BM cells were doubly stained with FITC-labeled anti-BP-1 (clone 6C3) and phycoerythrin-labeled anti-B220 (clone RA3-6B2; PharMingen, San Diego, CA) and analyzed using a FACScan from cytometer (Becton Dickinson, Mountain View, CA), as described previously (8). The lymphoid cells were gated according to forward scatter light and side scatter light. The percentage of BP-1+B220+ cells in this fraction was used as a quantitative parameter.

Linkage Analysis.

Genomic DNA was prepared from kidney according to standard protocols. All microsatellite primers were purchased from Research Genetics (Huntsville, AL), and PCRs were performed as described previously (7). A genome-wide scan was performed with back-crosses with high and low extremes of percentages of BM pre-B cells (34 mice each) and F2 mice with low and high extremes (16 mice each). They represented ∼10% of the population. They were genotyped for 56 marker loci distributed, on average, 25 cM apart, with a maximum distance of 34 cM between any two markers. This preliminary genome scan showed three possible linkages on chromosomes 3, 12, and 13, so that all individuals were genotyped at three to four loci on these chromosomes. Because the significant linkage was found at D3Mit317, further fine mapping was performed for nine loci on chromosome 3 and analyzed by the MapMaker-QTL software. All double recombinants were retyped and confirmed for their genotypes at these marker loci. LOD scores for marker loci on chromosomes 12 and 13 were <1.0 for 159 F2 mice, so further analysis was not performed.

Statistics.

Phenotype differences between sexes were analyzed by the unpaired t test. Two-way ANOVA was performed to detect differences between sex and origin of crosses and to detect the effect of sex on genotype.

To identify the genetic factors generating the BM pre-B-cell expansion, we examined BM pre-B cells in crosses between SL/Kh and NFS/N mice. NFS/N is an inbred strain with a low number of BM pre-B cells and a very low incidence of spontaneous lymphoma. BM pre-B cells were defined as BP-1+B220+ cells (Fig. 1), and their percentage among total BM lymphoid cells was used as a quantitative parameter throughout. The percentages of BM pre-B cells were 32.1 ± 9.2% in 34 SL/Kh mice and 3.1 ± 2.8% in 23 NFS mice at 4 weeks of age (P < 10−8). No significant difference was noted between the total BM cell yields of the two strains. The reciprocal F1 mice resulting from crosses between SL/Kh and NFS showed a value intermediate between those of the parents (Fig. 2). In back-crosses to SL/Kh and F2 cohorts, the percentage of BM pre-B cells showed an approximately normal distribution, indicating that it behaved as a quantitative trait, making it amenable to QTL analysis. Because a preliminary genome scan with high and low extremes of BM pre-B cells revealed a significant linkage on chromosome 3 (data not shown), all 334 back-cross mice to SL/Kh were genotyped for 9 loci on this chromosome. QTL analysis revealed a peak LOD score of 10.7, accounting for 16.4% of the overall phenotype variance at D3Mit319, 61.8 cM from the centromere of chromosome 3 (Fig. 3). Similar analysis for 159 F2 mice demonstrated an even higher LOD score of 22.7 at this region, accounting for 64% of overall phenotype variance in F2 mice (Fig. 4). Therefore, it is evident that a very high proportion of genetic variance is attributable to this locus. We named the QTL Bomb1.

We next evaluated the influence of Bomb1 on the percentage of BM pre-B cells in both F2 and back-cross mice with a two-way ANOVA (Table 1). It was clear that Bomb1 had a strong effect on the percentage of BM pre-B cells. Among the F2 mice, homozygotes for the SL/Kh allele (S/S) had the highest percentage of pre-B cells, the heterozygotes (S/N) had an intermediate percentage, and the homozygotes for the NFS allele (N/N) had the lowest percentage. Therefore, the manner of the effect can be described as additive. The additive nature of Bomb1 was further confirmed by comparing QTL models that were constrained to follow different models of inheritance. The results revealed that the additive model was the best fit, with no significant difference from the free inheritance model (Fig. 4).

The map position of Bomb1 was between D3Mit317 and D3Mit319, where LEF1 was mapped in the order D3Mit317-2.6 cM-LEF1-0.2 cM-D3Mit319.4LEF1 encodes a high mobility group protein transcription factor. In adults, it is expressed in pre-B and T lymphocytes but not in later stages of B cells (9). From the map position as well as its tissue distribution, LEF1 may be a promising candidate gene. One of its functions in lymphocytes is to regulate T-cell receptor α enhancer. Targeted inactivation of LEF1, however, has little effect on T-cell receptor α expression and T-cell differentiation because the function seems to be redundant with that of another related gene, T-cell factor 1(10). On the other hand, targeting of LEF1 abolishes the formation of organs that depend on epithelial-mesenchymal tissue interactions (11). The effect on the B-lineage has been not reported in detail because LEF1-deficient mice die immediately after birth. Our preliminary study on expression and cDNA sequences of LEF1 has failed to detect difference from NFS/N mice.

There was a significant sex difference in the percentage of BM pre-B cells in SL/Kh and F1 (Table 2): females had a higher level of pre-B cells than did males. We further analyzed the F1 and F2 cohorts by a two-way ANOVA to examine the effect of sex and the origin of cross on the number of pre-B cells. Sex had a significant effect on the tendency for females to have higher numbers of pre-B cells than males (in F1 hybrids, F = 23.17, P < 0.0001; in F2 hybrids, F = 7.4, P = 0.007). The origin of the cross had an effect on the F1 cohort (F = 5.69, P = 0.018) but no effect on the F2 cohort (F = 0.27, P = 0.6). When QTL analysis of chromosome 3 was performed separately for males and females, LOD scores were 16.5 in males and 5.6 in females of the F2 cohort and 6.7 in male back-cross mice and 4.5 in female back-cross mice. A two-way ANOVA indicated that sex had no significant effect on numbers of pre-B cells at the Bomb1 locus (F = 0.12, P = 0.72 in F2; F = 0.64, P = 0.43 in back-cross mice).

To examine the influence of the maternal effect on the level of pre-B cells, we had SL/Kh mothers foster-nurse NFS newborns or vice versa and examined BM pre-B cells at 4 weeks of age. All of these mice exhibited the same phenotype as their littermates nursed by their natural mother (data not shown). Therefore, we concluded that sex was the only additional factor other than the genetic factor that influenced the phenotypic variance.

Our previous study showed that the genetic requirements for pre-B lymphomas are endogenous ecotropic virus Emv11 and a dominant SL/Kh allele of Esl1 closely linked to the major histocompatibility locus in back-crosses to NFS/N (7). Of 145 mice resulting from back-crosses to NFS/N, 50 developed lymphomas during 18 months of observation (12). In this population, the SL/Kh allele at Bomb1 yielded a higher risk of lymphomas (P = 2.8 × 10−4). Therefore, Bomb1 plays an additional role in lymphoma predisposition. It remains unknown, however, whether the perturbance of pre-B differentiation by SL/Kh Bomb1 is due to a failure of apoptotic deletion of aberrant pre-B cells or simply due to expansion of a target cell population for transforming retrovirus. Further studies are underway to examine whether the pre-B expansion is a true prelymphoma lesion or represents one of the predisposing factors.

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

Supported by a Grant-in-Aid for Basic Research from The Ministry of Education, Culture, Sports and Science, Japan and by a grant from The Japan Owner’s Association.

            
3

The abbreviations used are: BM, bone marrow; QTL, quantitative trait locus; LOD, logarithm of odds; Bomb1, bone marrow pre-B-1; LEF1, lymphoid enhancer factor-1.

      
4

Mouse Genome Informatics, available at http://www.informatics.jax.org/. Accessed November 1998.

Fig. 1.

Flow cytometry of BM lymphoid cells doubly stained with phycoerythrin-labeled anti-B220 and FITC-labeled anti-BP1. The pre-B cells were defined as BP1+B220+ cells. Numbers, percentages of the cells within the respective quadrants. They were positive for CD19 and HSA but negative for IgM, CD5, CD43, CD29, CD44, intercellular adhesion molecule-1, leukocyte function-associated molecule-1, LECAM-1, and PD-1 (data not shown).

Fig. 1.

Flow cytometry of BM lymphoid cells doubly stained with phycoerythrin-labeled anti-B220 and FITC-labeled anti-BP1. The pre-B cells were defined as BP1+B220+ cells. Numbers, percentages of the cells within the respective quadrants. They were positive for CD19 and HSA but negative for IgM, CD5, CD43, CD29, CD44, intercellular adhesion molecule-1, leukocyte function-associated molecule-1, LECAM-1, and PD-1 (data not shown).

Close modal
Fig. 2.

Distribution of BM pre-B cells in SL/Kh and NFS mice and crosses between them at 4 weeks of age. •, individual mice; numbers, total numbers of mice in each group. Horizontal bar, mean value for each group. Data are from mice of both sexes.

Fig. 2.

Distribution of BM pre-B cells in SL/Kh and NFS mice and crosses between them at 4 weeks of age. •, individual mice; numbers, total numbers of mice in each group. Horizontal bar, mean value for each group. Data are from mice of both sexes.

Close modal
Fig. 3.

QTL analysis for the locus responsible for expansion of BM pre-B cells at 4 weeks of age on mouse chromosome 3. Distances between markers are in cM, calculated by Mapmaker/QTL program B. The LOD score is for SL/Kh × (NFS/N × SL/Kh) back-cross mice. The calculation was performed using data from both sexes.

Fig. 3.

QTL analysis for the locus responsible for expansion of BM pre-B cells at 4 weeks of age on mouse chromosome 3. Distances between markers are in cM, calculated by Mapmaker/QTL program B. The LOD score is for SL/Kh × (NFS/N × SL/Kh) back-cross mice. The calculation was performed using data from both sexes.

Close modal
Fig. 4.

QTL analysis for the F2 cohort. The free model was compared to the additive, dominant, and recessive models by a generalized likelihood ratio test at the QTL location. The best fit was the additive model. Data are from mice of both sexes.

Fig. 4.

QTL analysis for the F2 cohort. The free model was compared to the additive, dominant, and recessive models by a generalized likelihood ratio test at the QTL location. The best fit was the additive model. Data are from mice of both sexes.

Close modal
Table 1

Percentage of BM pre-B cells by genotype at the Bomb1-linked marker locus D3Mit319

Cross% BM pre-B cells, mean ± SD (no. of mice)FP
S/SaS/NaN/Na
S × NSF1 34.1 ± 13.3 (157) 24.8 ± 11.5 (174)  45.4 <0.0001 
SNF2 41.4 ± 8.9 (44) 22.2 ± 8.6 (86) 8.2 ± 5.3 (29) 73.1 <0.0001 
Cross% BM pre-B cells, mean ± SD (no. of mice)FP
S/SaS/NaN/Na
S × NSF1 34.1 ± 13.3 (157) 24.8 ± 11.5 (174)  45.4 <0.0001 
SNF2 41.4 ± 8.9 (44) 22.2 ± 8.6 (86) 8.2 ± 5.3 (29) 73.1 <0.0001 
a

Genotypes at D3Mit319.

Table 2

Effect of sex on BM pre-B cells

Mice% BM pre-B cells, mean ± SD (no. of mice)P
Both sexesFemaleMale
SL/Kh 32.1 ± 9.2 (34) 35.9 ± 4.3 (15) 29.1 ± 9.6 (19) 0.03 
NFS/N 3.1 ± 2.8 (23) 3.3 ± 2.9 (13) 2.8 ± 2.6 (10) 0.28 
SNF1 22.1 ± 5.6 (58) 25.6 ± 5.0 (25) 19.5 ± 4.5 (33) 0.00001 
NSF1 20.3 ± 5.3 (50) 21.8 ± 4.6 (26) 18.8 ± 5.6 (24) 0.04 
SNF2 24.9 ± 14.3 (43) 28.3 ± 12.9 (28) 18.4 ± 15.1 (15) 0.03 
NSF2 22.3 ± 11.3 (159) 23.1 ± 10.8 (70) 21.4 ± 11.6 (89) 0.37 
S × NSF1 29.6 ± 13.3 (334) 31.1 ± 12.6 (162) 28.3 ± 13.8 (171) 0.05 
Mice% BM pre-B cells, mean ± SD (no. of mice)P
Both sexesFemaleMale
SL/Kh 32.1 ± 9.2 (34) 35.9 ± 4.3 (15) 29.1 ± 9.6 (19) 0.03 
NFS/N 3.1 ± 2.8 (23) 3.3 ± 2.9 (13) 2.8 ± 2.6 (10) 0.28 
SNF1 22.1 ± 5.6 (58) 25.6 ± 5.0 (25) 19.5 ± 4.5 (33) 0.00001 
NSF1 20.3 ± 5.3 (50) 21.8 ± 4.6 (26) 18.8 ± 5.6 (24) 0.04 
SNF2 24.9 ± 14.3 (43) 28.3 ± 12.9 (28) 18.4 ± 15.1 (15) 0.03 
NSF2 22.3 ± 11.3 (159) 23.1 ± 10.8 (70) 21.4 ± 11.6 (89) 0.37 
S × NSF1 29.6 ± 13.3 (334) 31.1 ± 12.6 (162) 28.3 ± 13.8 (171) 0.05 

We thank Yukie Kanda for excellent technical assistance.

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