Experiment
A previous study (J:155722) demonstrated that an allelic variant of a Chr 17 QTL (Sst5) inherited from B6 mice determined resistance to tuberculosis infection. The goal of the current study was to narrow the region and identify the gene(s) influencing TB susceptibility/severity.
A panel of recombinant congenic mice were generated from the tuberculosis susceptible inbred mouse strain I/StSnEgYCit (I/St) and the tuberculosis resistant strain C57BL/6JCit (B6). Genomic regions covering the vicinity of the H2 complex from parental I/St-H2j mice were transferred onto the B6-H2b genetic background in successive backcross generations. At generation backcross one (N2), simultaneous selection for the presence of two traits was applied: TB-susceptible phenotype and Chr 17 markers of I/St origin. At generation N10-11 more than 40 B6.I-H2 recombinant congenic strains on the B6 background carrying different, partly-overlapping, genomic regions of the extended H2j haplotype (Chr17:8.44-65.34 Mb) were generated.
Figure 1 displays the most informative B6.I recombinant congenic strains whose phenotyping and genotyping allowed the region of interest to be narrowed to an interval between (rs13482956 and H2-Ea) 33.77-34.34 Mb. Mice of all strains that inherited this region from I/St ancestors were significantly more susceptible to infection than those bearing B6 alleles. MGI curators have labeled the identified interval as QTL Sts6, susceptibility to tuberculosis 6. The previously identified QTL Sst5, also on Chr 17, was mapped using a different mapping population (C3H.B6 x C57BL/6)F2.
The identified region, 33.77-34.34 Mb, contained 36 protein-coding genes according to http://www.ensembl.org (GRCm38). The protein coding regions of all 36 I/St originated genes were cloned and sequenced (GenBank accession numbers KJ650201-KJ650234). All but 7 genes appeared to be highly polymorphic.
Several more crosses were performed to search for new recombination events inside the 33.77-34.34 Mb interval. The F2 progeny of B6.I-(D17Mit13-D17Mit16)/Cit x C57BL/6JCit mice were used to develop a set of new congenic strains. Standard genotyping identified the point of recombination between D17Mit21 (34.24 Mb) and D17Mit22 (34.33 Mb) in two new recombinant strains, B6.I-249.1.15.100 (B6.I-100) and B6.I-249.1.15.139 (B6.I-139). After aerosol challenge survival of B6.I-100 mice differed significantly from resistant B6.I-139 mice, p<0.001. Phenotypic differences were confirmed by evaluation of cachexia dynamics and by assessment of mycobacterial loads in the lungs at weeks 4 and 10 post challenge. The genomic region between D17Mit21 and D17Mit22 contained only 5 protein coding genes, 2 linc RNA genes and no micro-RNA genes.
Gene sequencing revealed that both strains carried the b allele of H2-Ob and the j allele of H2-Aa, but differed at the H2-Ab1 gene. In B6.I-139 mice, the whole polymorphic part of the H2-Ab1 gene encoding the extracellular functional domain of the molecule was of H2b origin: only substitutions W222R in the connecting peptide and H249Y in the cytoplasmic domain were inherited from the H2j haplotype. In contrast, in B6.I-100 mice the polymorphic part of the H2-Ab1 gene was identical with the H2j haplotype, except for a single substitution N29D.
Recombinant breakpoints occurred in different sites of the H2-Ab1 gene providing polymorphic variations in the domain B1 of the AB-chain; variations sufficient to produce different TB relevant phenotypes. The more susceptible B6.I-100 strain demonstrated shorter survival time, more rapid body weight loss, higher mycobacterial loads in the lungs and more severe lung histopathology compared to the more resistant B6.I-139 strain.
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