|Coordinate||106,224,139 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||toll-like receptor 9|
|Chromosomal Location||106,222,598-106,226,883 bp (+)|
|MGI Phenotype||Mice homozygous for a knock-out allele exhibit impaired immune system response to LPS, CpG, and Leishmania bazillensis infection.|
|Amino Acid Change||Asparagine changed to Tyrosine|
|Institutional Source||Beutler Lab|
N210Y in Ensembl: ENSMUSP00000082207 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||immune system, TLR signaling defect: type I IFN production by pDC|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Sperm, gDNA|
|Last Updated||2017-04-11 2:53 PM by Katherine Timer|
|Record Created||2010-06-18 3:33 PM by Amanda L. Blasius|
The Meager phenotype was identified in a screen of ENU-mutagenized homozygous G3 mice looking for reduced type I interferon (IFN) responses to CpG DNA challenge in vivo (Figure 1).
|Nature of Mutation|
The Meager mutation was mapped to Chromosome 9 by outcrossing the index mouse to the closely related C57BL/10J strain and intercrossing F1 animals. Progeny were tested for in vivo type I interferon responses (Figure 2) and the DNA samples from phenotypically wild type and mutant animals were pooled separately and analyzed by bulk segregation analysis (BSA) using a panel of 124 single nucleotide polymorphisms (SNPs) (1). Sequencing of the candidate gene Tlr9 on Chromosome 9 found an A to T transversion at position 734 of the Tlr9 transcript using Genbank record NM_031178.2, in exon 2 of 2 total exons (Figure 3).
The mutated nucleotide is indicated in red lettering, and causes an asparagine to tyrosine substitution at amino acid 210 of the TLR9 protein.
The Meager mutation results in an asparagine to tyrosine amino acid change at position 210 of the TLR9 protein, which lies in the predicted sixth LRR module of the TLR9 ectodomain.
Please see the record for CpG1 for information about Tlr9.
The Meager mutation substitutes tyrosine for asparagine at position 210 of the TLR9 protein, which lies in the predicted sixth LRR module of the TLR9 ectodomain and is conserved in human TLR9. This residue is located just after the invariant tenth asparagine residue present in all LRR motifs (XLXXLXLXXN), which folds into a β-strand. No tertiary structural data presently exist for TLR9, making it difficult to hypothesize how the Meager mutation could affect either ligand binding or receptor dimerization. Recently, the crystal structure of the related TLR3 heterodimer bound to double-stranded (ds) RNA has been elucidated (see record for CpG1). The ligand-bound TLR3 heterodimer forms an M-shape with the dsRNA binding to the concave surfaces of the TLR3 heterodimer at two locations on each ectodomain (2). It has been hypothesized that TLR7, 8 and 9 ligands may also bind to the concave surface of the ectodomain at a site made up by insertions at LRR 2, 5, 8 and 11 (3). The Meager mutation might somehow disrupt ligand binding and/or receptor dimerization, or destroy proper folding or localization of the receptor.
Only homozygous Meager mice have been tested in the in vivo CpG screen, but Tlr9 mutations are known to behave semidominantly in other assays. The Meager mutation is thus tentatively classified as semidominant.
|Primers||Primers cannot be located by automatic search.|
Meager genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
meager(F): 5’- TGGCTGTTCCTGAAGTCTGTACCC -3’
meager(R): 5’- AGATGCCGTTCATGTTCAGCTCC -3’
1) 95°C 2:00
2) 95°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C 8
Primers for sequencing
meager_seq(F): 5'- GTTTCTCTGCGGCAGCATC -3'
meager_seq(R): 5'- CTTGACAATGAGGTTATAGGACACC -3'
The following sequence of 985 nucleotides (NCBI Mouse Genome Build 37.1, Chromosome 9, bases 106,125,981 to 106,126,965) is amplified:
tggctgtt cctgaagtct gtaccccgtt tctctgcggc agcatcctgc tccaacatca cccgcctctc cttgatctcc aaccgtatcc accacctgca caactctgac ttcgtccacc tgtccaacct gcggcagctg aacctcaagt ggaactgtcc acccactggc cttagccccc tgcacttctc ttgccacatg accattgagc ccagaacctt cctggctatg cgtacactgg aggagctgaa cttgagctat aatggtatca ccactgtgcc ccgactgccc agctccctgg tgaatctgag cctgagccac accaacatcc tggttctaga tgctaacagc ctcgccggcc tatacagcct gcgcgttctc ttcatggacg ggaactgcta ctacaagaac ccctgcacag gagcggtgaa ggtgacccca ggcgccctcc tgggcctgag caatctcacc catctgtctc tgaagtataa caacctcaca aaggtgcccc gccaactgcc ccccagcctg gagtacctcc tggtgtccta taacctcatt gtcaagctgg ggcctgaaga cctggccaat ctgacctccc ttcgagtact tgatgtgggt gggaattgcc gtcgctgtga ccatgccccc aatccctgta tagaatgtgg ccaaaagtcc ctccacctgc accctgagac cttccatcac ctgagccatc tggaaggcct ggtgctgaag gacagctctc tccatacact gaactcttcc tggttccaag gtctggtcaa cctctcggtg ctggacctaa gcgagaactt tctctatgaa agcatcaccc acaccaatgc ctttcagaac ctaacccgcc tgcgcaagct caacctgtcc ttcaattacc gcaagaaggt atcctttgcc cgcctccacc tggcaagttc ctttaagaac ctggtgtcac tgcaggagct gaacatgaac ggcatct
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is indicated in red.
1. Xia, Y., Won, S., Du, X., Lin, P., Ross, C., La Vine, D., Wiltshire, S., Leiva, G., Vidal, S. M., Whittle, B., Goodnow, C. C., Koziol, J., Moresco, E. M., and Beutler, B. (2010) Bulk Segregation Mapping of Mutations in Closely Related Strains of Mice. Genetics 186, 1139-1146.
2. Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M., and Davies, D. R. (2008) Structural basis of toll-like receptor 3 signaling with double-stranded RNA, Science 320, 379-381.
|Science Writers||Nora G. Smart|
|Authors||Amanda L. Blasius, Bruce Beutler|