|Coordinate||129,555,640 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||lymphocyte protein tyrosine kinase|
|Chromosomal Location||129,548,349-129,573,641 bp (-)|
|MGI Phenotype||Mice homozygous for mutations of this gene exhibit thymic atrophy with reduced numbers of peripheral T cells. Null mutants have few double positive and no mature single positive (SP) thymocytes. A hypomorph has decreased expression of CD3epsilon chain on SP thymocytes, whose numbers are reduced.|
|Amino Acid Change||Glutamic Acid changed to Glycine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000125777]|
AA Change: E299G
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||decrease in CD4:CD8, decrease in CD4+ T cells, decrease in CD4+ T cells in CD3+ T cells, decrease in CD8+ T cells, decrease in T cells, DSS: resistant day 10, increase in B:T cells, increase in CD44 MFI in CD4, increase in CD44 MFI in CD8, increase in CD8+ T cells in CD3+ T cells, T-dependent humoral response defect- decreased antibody response to OVA+ alum immunization, T-dependent humoral response defect- decreased antibody response to rSFV|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice|
|Last Updated||03/03/2017 10:59 AM by External Program|
|Record Created||08/07/2013 1:38 PM by Emre Turer|
he stromberg phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R0480, some of which showed an increase in the B:T cell ratio (Figure 1) due to a decreased frequency of total T cells (Figure 2). Some mice also showed a decrease in the CD4+ to CD8+ T cell ratio (Figure 3) caused by a diminished frequency of CD4+ T cells (Figure 4) and CD4+ T cells in CD3+ T cells (Figure 5) coupled with lesser diminution of CD8+ T cells (Figure 6) and an increase in the frequency of CD8+ T cells in CD3+ T cells (Figure 7). CD44 expression was increased on both CD4+ and CD8+ T cells (Figure 8 and 9, respectively). Some mice showed diminished T-dependent IgG responses to both aluminum hydroxide (alum)-emulsified ovalbumin (OVA) (Figure 10) and recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal) (Figure 11).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 114 mutations. Three mutations (in Ago4, Lck, and Cnr2) shared equal linkage to all of the above anomalies by continuous variable mapping. The mutation in Lck is presumed to be causative as it shares immune phenotypes with other alleles of Lck (see MGI and the record for iconoclast). The Lck mutation is A to G transition at base pair 129,555,640 (v38) on chromosome 4, equivalent to base pair 18,002 in the GenBank genomic region NC_000070 encoding Lck. The strongest association was found with a recessive model of linkage to the normalized T-dependent antibody response to rSFV-β-gal, wherein 10 variant homozygotes departed phenotypically from 8 homozygous reference mice and 16 heterozygous mice with a P value of 3.568 x 10-18 (Figure 12).
The mutation corresponds to residue 953 in the NM_001162432 mRNA sequence in exon 9 of 13 total exons, or at position 995 bp of the NM_001162433 mRNA sequence in exon 9 of 13 total exons, or at position 1,050 of the NM_010693 mRNA sequence in exon 8 of 12 total exons.
Genomic numbering corresponds to NC_000070. The mutated nucleotide is indicated in red lettering and results in a glutamic acid to glycine substitution at position 299 (E299G) in the Lck protein isoform encoded by NM_001162432 and a glutamic acid to glycine substitution at position 288 (E288G) in the Lck protein isoforms encoded by NM_001162433 and NM_010693.
Lck encodes lymphocyte protein tyrosine kinase (Lck), a member of the Src family of nonreceptor tyrosine kinases (Figure 13). The stromberg mutation lies in the N-lobe of the Lck kinase domain (amino acids 238-496), within helix αC [Figure 14; (1-4)]. The effect of the mutation on kinase activity, expression level, or localization has not been tested.
Please see the record iconoclast for information about Lck.
Lck functions in one of the first steps in T cell receptor (TCR) signaling, in which it phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMS) present on the CD3 heterodimer (CD3εγ or CD3εδ) and CD3ζ chains of the TCR. This event is necessary for the propagation of signals from the TCR. The stromberg mutation lies in the N-lobe of the Lck kinase domain, which may alter the kinase activity.
stromberg(F):5'- AGGTAAAAGCGCCTTCCTTCCCTG -3'
stromberg(R):5'- AAAGTTGAGCCGTCCTTGCCAG -3'
stromberg_seq(F):5'- CAAAAGGTAGTGGGACCACA -3'
stromberg_seq(R):5'- TTCCCAGGGAAACACTGAAGTTG -3'
Stromberg 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.
Stromberg(F): 5’- AGGTAAAAGCGCCTTCCTTCCCTG-3’
Stromberg(R): 5’- AAAGTTGAGCCGTCCTTGCCAG-3’
Stromberg_seq(F): 5’- CAAAAGGTAGTGGGACCACA-3’
Stromberg_seq(R): 5’- TTCCCAGGGAAACACTGAAGTTG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C ∞
The following sequence of 532 nucleotides is amplified (Chr. 4: 129555452- 129555983, GRCm38; NCBI RefSeq NC_000070):
aggtaaaagc gccttccttc cctgggtcca aaaggtagtg ggaccacaat agcgccctca
agcggctgga catagcaggg cacccaccgt tctccatgta ttccgtgatg atgtagatgg
gttcctgggt gaccactgca taaagccgga ctagccgcgg gtgctgcagc tgcttcatga
ggttagcctc agccaggaag gcgtcggggg acatgctccc ttgtttcaga ctcttcaccg
ccaccttcgt gtgtccgttg tagtaccctg atggggtacg ggtggagaag ttacagaggt
caagatcctg acgaactagg cagccatcct gaataggcca gtgtgagagg aagctcacat
tctctctcct ctttccaatc agtcccgagg gtcacactca cccatccaca cttccccgaa
ctggccagct cccagccgct ccaccaactt cagtgtttcc ctgggaactt cccattcgtc
ctcccaccat ggtttctggg gcttctgggt ctggcaagga cggctcaact tt
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (T>C, Chr. + strand (shown); A>G, sense strand).
1. Xu, W., Harrison, S. C., and Eck, M. J. (1997) Three-Dimensional Structure of the Tyrosine Kinase c-Src. Nature. 385, 595-602.
2. Sicheri, F., Moarefi, I., and Kuriyan, J. (1997) Crystal Structure of the Src Family Tyrosine Kinase Hck. Nature. 385, 602-609.
3. Sicheri, F., and Kuriyan, J. (1997) Structures of Src-Family Tyrosine Kinases. Curr Opin Struct Biol. 7, 777-785.
|Science Writers||Anne Murray|
|Authors||Emre Turer, Kuan-Wen Wang, Jin Huk Choi, Ming Zeng, Bruce Beutler|