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{"project":"2_test","denotations":[{"id":"17244351-15529344-4361130","span":{"begin":337,"end":339},"obj":"15529344"},{"id":"17244351-15529344-4361131","span":{"begin":864,"end":866},"obj":"15529344"},{"id":"17244351-10754334-4361132","span":{"begin":867,"end":869},"obj":"10754334"},{"id":"17244351-2563713-4361133","span":{"begin":1522,"end":1524},"obj":"2563713"},{"id":"17244351-12594249-4361134","span":{"begin":2120,"end":2122},"obj":"12594249"},{"id":"17244351-10453039-4361135","span":{"begin":2230,"end":2232},"obj":"10453039"},{"id":"17244351-11046062-4361136","span":{"begin":2386,"end":2388},"obj":"11046062"},{"id":"17244351-9724775-4361137","span":{"begin":2857,"end":2858},"obj":"9724775"},{"id":"17244351-12687532-4361138","span":{"begin":2859,"end":2860},"obj":"12687532"},{"id":"17244351-2563713-4361139","span":{"begin":7364,"end":7366},"obj":"2563713"}],"text":"Results\n\nSmall-effect QTL of CIA in (DBA/1 × FVB/N) F2 progeny\nIn a previous study, we carried out a genome screen to identify QTL controlling CIA susceptibility in (DBA/1 × FVB/N) F2 progeny. For the phenotype of arthritis severity, only one QTL, Cia2, was identified, with a highly significant logarithm of the odds (LOD) score of 12 [10]. However, Cia2 contributed to only 16% of the phenotype variations, indicating that there should be some small-effect QTL whose contributions to CIA were not big enough to reach the significant threshold value of linkage. To identify these potential small-effect QTL, we reanalyzed the data using a lower threshold value of linkage (P = 0.05). We reasoned that since the main candidate gene of Cia2, complement component C5 (Hc), was proven to be essential for CIA development and because the FVB/N strain is C5 deficient [10,25], some small-effect QTL might be masked by Cia2. To exclude the masking effect of C5, we performed linkage analysis with 3 datasets, the first containing all 290 F2 progeny, the second 77 C5+/+ F2 progeny and the third 133 C5+/- F2 progeny. Eight genomic regions were linked to the phenotype of CIA severity (loci 1 to 8, Table 2), with P values varying between 0.043 and 0.003. These eight small-effect QTL were located on chromosomes 5, 6, 7, 10, 11, 16, and 17. Five loci were identified in at least two datasets. Of the eight loci, five had DBA/1 as the susceptibility allele, and three had FVB/N as the susceptibility allele.\nLander and Botstein [26] suggested a LOD score of between 2 and 3 to ensure an overall false positive rate of 5%, which means that using a lower threshold value will prevent false negative QTL at the expense of increasing false positive QTL. Being aware of this, we examined these genomic regions to search whether they, or their syntenic genomic regions on the human genome, have been previously linked to arthritis. Four small-effect QTL overlapped with arthritis QTL on the mouse genome identified previously. Locus 1 and 2 overlap with Cia13 and Cia14, which control severity of CIA in (DBA/1 × BALB/C) F2 progeny [27]. Locus 5 located on chromosome 10 overlaps with Cia8, which was identified in (DBA/1 × B10.Q) F2 progeny [28]. Locus 6 overlaps with Pgia7, which controls susceptibility to proteoglycan-induced arthritis (PGIA) and was identified in (BALB/C × DBA/2) F2 progeny [29]. The syntenic genomic regions of five small-effect QTL on the human genome have been reported to be linked to RA. These are genomic regions 22q11 and 12p13-q24 on chromosome 22 and 12 (the counterparts of locus 2), 12p13-pter on chromosome 12 (the counterpart of locus 3), 21q22-qter and 10q22-23 on chromosome 21 and 10 (the counterparts of locus 5), 17q21-25 on chromosome 17 (the counterpart of locus 6) and 3q29-qter on chromosome 3 (the counterpart of locus 7) [2,4].\n\nStrain-specific differentially expressed genes\nWe detected the gene expression profiles using three mice per group, which is a small number. Being aware of the importance of data reproducibility, we determined the coefficient of variation (CV) to measure data variability. The CV for each gene on the chip and the mean CV for the entire probe set were calculated. The mean CV ranged between 18.4% and 25.8% for all experimental groups, and this relatively low CV indicated that these data could be used for further analysis (Table 1).\nTo search for strain-specific differentially expressed genes, we performed comparisons of gene expression between the DBA/1 and FVB/N strains at all four phases of CIA, including NC, PI, OA and CA. For the naive mice without immunisation, 361 genes were differentially expressed between the two strains. On day 10 after immunisation, when both strains did not show any sign of the disease, 141 genes were differentially expressed. After DBA/1 mice developed CIA, 184 and 85 differentially expressed genes were identified between these two strains at the onset and chronic phases, respectively. When the lists of the differentially expressed genes at the four phases were merged and overlapping genes were excluded, 509 genes were identified (Additional file 1). Twenty-one genes consistently showed differential expression between the two strains at all phases. Besides these 21 genes, only 3 additional genes were strain-specific differentially expressed during the 3 phases after CIA induction (PI, OA and CA; Figure 1).\n\nDisease-specific differentially expressed genes\nTo identify the disease-specific differentially expressed genes in CIA, we detected the genes that were differentially expressed in LNs during CIA in the susceptible strain. Three experimental conditions, PI, OA and CA, were compared with the NC group. On day 10 after immunisation, 102 genes were differentially expressed – most of them were upregulated (78 out of 102) – while at the onset phase of the disease, only 26 genes were differentially expressed. At the chronic phase of the disease, 184 differentially expressed genes were identified, with 156 downregulated genes. Only one gene was differentially expressed at all three phases of CIA. Besides this gene, five, one and six differentially expressed genes were shared by PI with OA, PI with CA and OA with CA, respectively (Figure 2a). Taken together, 310 disease-specific differentially expressed genes were differentially regulated during CIA in DBA/1 mice (Additional file 2).\nTo further characterise the gene expression pattern during CIA, we performed hierarchical cluster analysis for these 310 genes. Six gene clusters were identified (clusters I to VI, Figure 2b), each with a distinct gene expression pattern during CIA. Cluster I contains 16 genes, representing genes that were upregulated after induction of CIA. The expression of these genes reached a peak at the onset phase of the disease and functional clustering results revealed that they are related to the immune response. Cluster II contains 12 genes whose expression was gradually upregulated and reached a peak at the chronic phase of CIA. These genes are mainly related to the immune response, organelle membrane and extracellular region and space. Cluster III contains 78 genes that were only upregulated at the PI phase. These genes are related to the intercellular junction. More than half of the genes (156 of 310) belong to cluster IV and represent genes specifically downregultaed at the chronic phase. These genes are functionally related to lymphocyte proliferation, T cell activation, protein binding as well as the notch signal pathway. Cluster V contains eight genes downregulated at the PI phase. Cluster VI contains 18 genes downregulated at the OA phase. The GO term classification showed no functional cluster that was significantly enriched in these two gene clusters.\n\nCandidate genes for the small-effect QTL of CIA\nTo identify candidate susceptibility genes for the CIA small-effect QTL, we compared the list of strain-specific differentially expressed genes with the list of disease-specific differentially expressed genes; 117 genes were shared by both lists (Additional file 3). Figure 3 visualises positions of the 117 genes retrieved from Ensembl [30] in relation to the 8 small-effect QTL. The eight loci were located on 7 chromosomes, 5, 6, 7, 10, 11, 16 and 17. Since the confidence intervals of QTL in F2 progeny are around 20 cM [26], we used 40 Mb as the confidence intervals for all loci. Twenty-one genes were located in the confidence intervals of six of the eight QTL. We located 5, 4, 2, 1, 3 and 6 potential candidate genes within the confidence intervals of loci 1, 2, 3, 5, 6 and 8, respectively, while no candidate gene was identified for loci 4 and 7. Table 3 summarises the 21 candidate genes identified in this study. Two genes, hspa1a and Oas1a, were upregulated at the OA phase of CIA and Oas1a was also upregulated at the PI phase. Except for these two genes, all other 19 genes were downregulated at the chronic phase of CIA. All genes, except hspa1a, showed expression differences between the two strains at the NC phase. Five genes were differentially expressed at all phases of CIA, including H2-Q10, Mapk14, Pscd1, Kpnb1 and Wdr1. Among these five genes, H2-Q10 had a consistently higher expression in the DBA/1 than the FVB/N strain in all CIA phases, while the other four genes had a higher expression in the DBA/1 strain at the early stages, including NC, PI and OA, but a lower expression at the chronic phase. GO term classification analysis revealed that the functional cluster of protein kinase cascade was significantly enriched in the 21 candidate genes. This functional cluster contained four genes, including Mapk14, Mapk8ip3, Stat5a and Gna12."}

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PMC:1860061 / 10336-19046 JSON TXT