Experiment
The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Knj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of myeloperoxidase specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) and marked leucocytosis. The current study was performed to identify the specific populations of leucocytes associated with CrGN and susceptibility loci for pathogenic leucocytosis.
Four hundred and twenty female (C57BL/6 SCG/Knj)F2 (BxSF2) intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were examined histopathologically.
PB was obtained from periorbital sinus every 4 weeks. For monitoring the onset of glomerulonephritis (GN), proteinuria and haematuria were tested. Mice were killed at 24 weeks of age or when proteinuria was more than 200 mg/dl and/or haematuria was more than 1+. At autopsy, kidneys were examined histopathologically. GN was expressed as percentage of glomeruli with endocapillary proliferation and/or mesangial proliferation and/or mesangial sclerosis. The crescent formation was expressed as numbers of glomeruli with crescents among 120 glomeruli. Vasculitis was expressed as numbers of small vessels with granulomatous vasculitis in four independent kidney sections.
A mapping panel was constructed using BXSF2 with the modification of adding seven markers
on the middle of chromosome 1 (D1MIT490, D1MIT492,
D1MIT387, D1MIT91, D1MIT139, D1MIT536 and D1MIT30). Genomic DNA of mice were extracted from tail samples. Genotypes were determined by polymerase chain reaction (PCR) using selected simple sequence-length polymorphism markers. Genotypes of Fas were represented by those of D19MIT87 because of their adjacent positions on chromosome 19.
Comparison of leucocyte counts in PB among B6, BxSF1, healthy BxSF2 and diseased BxSF2 mice was performed with analysis of variance (ANOVA). Associations between
histopathological traits and leucocyte counts in BxSF2 mice were determined by correlation coefficients with P-values derived from Fishers transformation.
The linkage map for the BxSF2 was created using MapManager QTX b20. ( http://www
.mapmanager.org/). Interval mapping for QTL detection was performed using MapManager QTX in a free regression model. Likelihood ratio statistics were converted to conventional
base-10 logarithm of odds (LOD) scores. To establish suggestive and significant threshold values, permutation tests were performed using MapManager QTX with 1000 permutations of the data.
A genome-wide scan was performed to search non-Fas QTL for leucocytosis using MapManager QTX software.
Three significant susceptibility QTL were identified Table 3, Fig 5:
QTL Leusq1 (leucocytosis susceptibility QTL 1) mapped to Chromosome 1 peaking with marker D1Mit15, LOD=6.5, p=0.0001. Leusq1 was associated with an increase in whole and conventional dendritic cells in peripheral blood in an interval of Chr 1 between D1Mit14 and D1Mit15 that accounted for 7% of trait variance.
QTL Leusq2 (leucocytosis susceptibility QTL 2) mapped to Chromosome 1 peaking with marker D1Mit387, LOD=5.1, p=0.0001. Leusq2 was associated with an increase in whole and conventional dendritic cells in peripheral blood in an interval of Chr 1 between D1Mit134 and D1Mit91 that accounted for 6% of trait variance.
Both of the Chr 1 QTL were derived from SCG/Knj alleles and were inherited in a recessive manner; both QTL predominantly controlled the frequency of conventional dendritic cells in peripheral blood. Leusq1 and Leusq2 overlap with previously reported [J:108420] GN-controlling SCG/Knj loci Scgq1 and Scgq2, spontaneous crescentic glomerulonephritis QTL 1 and 2.
QTL Leusq3 (leucocytosis susceptibility QTL 3) was identified on Chromosome 17 peaking with marker D17Mit21, LOD=4.5, p=0.005. Leusq3 was associated with an increase in plasmacytoid dendritic cells in an interval mapping of Chr 15 between D17Mit246 and D17Mit64 that accounted from 5% of trait variance. Leusq3 was also derived from SCG/Knj alleles, inherited recessively.
Three additional QTL were identified with lod support intervals and peak markers resembling those of Scg-1/Leusq1 and Scg-2/Leusq2. All three loci were also derived from SCG/Knj alleles, inherited recessively. Fig 6a,b:
QTL Leusq4 (leucocytosis susceptibility QTL 4) mapped to Chromosome 1 between markers D1Mit14 and Fcgr2b, LOD=5.5, and was associated with an increase of granulocytes (p<0.0001) and macrophages/monocytes (p<0.005) in peripheral blood cells.
QTL Leusq5 (leucocytosis susceptibility QTL 5) mapped to Chromosome 1 between markers D1Mit11 and D1Mit91 peaking with marker D1Mit387, LOD=4.5, and was associated with an increase of granulocytes (p<0.005) and macrophages/monocytes (p<0.0005) in peripheral blood cells.
QTL Leusq6 (leucocytosis susceptibility QTL 6) mapped to Chromosome 17 peaking with marker D17Mit21, LOD=4.7, and was associated with an increase of granulocytes (p<0.0005) in peripheral blood cells.
A systematic pairwise genome scan was performed using MapManager QTX. Significant interactions between Fas and Leusq1 and Leusq4 (D1Mit15) were detected for cDCs, granulocytes and macrophages/monocytes.
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