Phenotypic Mutation 'elektra2'

• Allele: elektra2
• Mutation Type: missense
• Chromosome: 11
• Coordinate: 82,960,390 bp (GRCm39)
• Base Change: A ⇒ T (forward strand)
• Gene: Slfn2
• Gene Name: schlafen 2
• Synonym(s): Shlf2
• Chromosomal Location: 82,955,938-82,961,504 bp (+) (GRCm39)
• MGI Phenotype: PHENOTYPE: Mice homozygous for an ENU-induced allele exhibit increased susceptibility to bacterial and viral infections, reduced T cell numbers, decreased T cell proliferation, and increased apoptosis of activated T cells. [provided by MGI curators]
• Mapped: Yes
• Limits of the Critical Region: 83065112 - 83070678 bp
• Amino Acid Change: Histidine changed to Leucine
• Institutional Source: Beutler Lab
• Gene Model: predicted gene model for protein(s): [ENSMUSP00000035562]
• AlphaFold: Q9Z0I6
• SMART Domains: Protein: ENSMUSP00000035562; Gene: ENSMUSG00000072620; AA Change: H123L

Domain	Start	End	E-Value	Type
low complexity region	162	172	N/A	INTRINSIC
Pfam:AlbA_2	231	365	4.5e-12	PFAM

• Predicted Effect: probably benign ; PolyPhen 2 Score 0.045 (Sensitivity: 0.94; Specificity: 0.83)
  (Using ENSMUST00000038038)
• Meta Mutation Damage Score: 0.0898
• Is this an essential gene?: Probably nonessential (E-score: 0.049)
• Phenotypic Category: Autosomal Recessive
• Mode of Inheritance: Autosomal Recessive
• Last Updated: 2019-09-04 9:44 PM by Anne Murray
• Record Created: 2015-11-12 7:52 PM by Jin Huk Choi
• Record Posted: 2016-06-03
• Science Writers: Anne Murray
• Illustrators: Peter Jurek
• Authors: Jin Huk Choi, James Butler, Bruce Beutler

Accession Number

NCBI RefSeq: NM_0011408; MGI:1313258

Alleles Listed at MGI

All Mutations and Alleles(2) : Chemically induced (ENU)(1) Targeted(1)

Phenotypic Description

Figure 1. Homozygous elektra2 mice exhibit diminished T-dependent IgG responses to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal). IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The elektra2 phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R3437, some of which showed a diminished T-dependent antibody response to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal) (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the diminished T-dependent antibody response to rSFV-β-gal using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 48 mutations (X-axis) identified in the G1 male of pedigree R3437. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 48 mutations. The reduced T-dependent antibody response to rSFV-β-gal phenotype was linked by continuous variable mapping to a mutation in Slfn2:  an A to T transversion at base pair 83,069,564 (v38) on chromosome 11, or base pair 4,453 in the GenBank genomic region NC_000077 for the Slfn2 gene. Linkage was found with a recessive model of inheritance (P = 4.673 x 10^-5), wherein four variant homozygotes departed phenotypically from 11 homozygous reference mice and 10 heterozygous mice (Figure 2).  

The mutation corresponds to residue 550 in the mRNA sequence NM_0011408 within exon 2 of 2 total exons.

534 CCCTTTGTCCAGTGGCACCTGGACTTCACGGAG 118 -P--F--V--Q--W--H--L--D--F--T--E-

The mutated nucleotide is indicated in red. The mutation results in a histidine (H) to leucine (L) substitution at position 123 (H123L) in the Slfn2 protein, and is strongly predicted by PolyPhen-2 to be benign (score = 0.045) (1).

Protein Prediction

Figure 3. Domain structure of Slfn2. The COG2685 domain partially overlaps the divergent AA domain. No other domains have been identified in SLFN2. The position of the elektra2 mutation is indicated.

Schlafen2 is one of ten Schlafen (Slfn) protein family members that exist in mice and are thought to regulate thymocyte maturation and T cell activation. Slfns 1-4 contain a core region of 120 residues that are nearly identical, with divergent sequences at the N terminus. Slfn proteins contain several motifs identified based on sequence similarity to other proteins (Figure 3). All Slfns possess a region, designated COG2685 (NCBI Conserved Domain Database), with similarity to a domain present in several ATP-dependent DNA helicases, transcriptional regulators, as well as several hypothetical proteins from both prokaryotes and eukaryotes (2). The domain, of unknown function but predicted to contain a helix-turn-helix DNA binding motif, has been referred to as the “Slfn box” (3). In Slfn2, this domain is positioned between amino acids 258-280. Adjacent and overlapping the COG2685 domain, Slfns contain a divergent AAA domain, which is related to the AAA (ATPases associated with various cellular activities) domain present in proteins of the AAA-containing family (4).

The elektra2 mutation, which converts histidine 123 to leucine, is found in a region of Slfn2 for which no functional domains have yet been identified.

Please see the elektra record for more information about Slfn2.

Putative Mechanism

The function of the SLFN protein family has been investigated in several studies, but so far relatively little is known. Initial reports suggested that Slfns 1-3 have growth inhibitory properties. Studies of elektra mice demonstrate that a Slfn2 mutation causes immunodeficiency as a consequence of T cell and monocyte apoptosis secondary to a semi-activated phenotype (5). The semi-activated state in Slfn2^{elektra/elektra} T cells stems from a loss of cellular quiescence (5). Slfn2 is proposed to maintain the quiescent state by promoting the expression of quiescence genes and/or repression of genes required for proliferation/differentiation. The failure of mature homozygous elektra T cells to engage the anti-apoptotic machinery of the cell stems from loss of quiescence and acquisition of a semi-activated phenotype by naïve elektra T cells, and is not a consequence of impaired survival signaling or activation by a lymphopenic environment (5). 

The activation of several pathways, including the NF-κB, NFAT, AKT, ERK1/2, JNK, and p38-MAP kinase pathways, regulates T cell survival. In the case of homozygous elektra mutants, we presume that as a result of a loss of quiescence, the balance between these pathways is altered to favor apoptosis rather than expansion. The enhancement of p38-MAPK and JNK phosphorylation observed upon TCR stimulation of elektra T cells, and the fact that inflammatory monocytes (of the myeloid lineage) also undergo apoptosis upon activation, suggest that the proliferative signals negatively regulated by Slfn2 may be distinct from the TCR signaling pathways that lead to the development of activated T cell effector functions and full T cell activation. Slfn2 is required for normal proliferation of CD8⁺ T cells in response to TCR stimulation, PMA/ionomycin treatment, and LCMV infection. The elektra mice exhibited delayed production of antibodies in response to MCMV infection.

The phenotype of the elektra2 mice indicates some loss-of-function of Slfn2^elektra2. However, other immune cell phenotypes observed in the elektra strain were not observed or tested in elektra2, indicating that Slfn2^elektra2 may retain some function.

Primers

PCR Primer
elektra2_pcr_F: TTCTCACCTCAGAAAACAGGAG
elektra2_pcr_R: TTCCACTTCAGGGAAGGCTATG

Sequencing Primer
elektra2_seq_F: GGAGAATGCAAACATCTCTCTAGCTG
elektra2_seq_R: AACTCTGGTCTGACACAGGG

Genotyping

PCR program

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 hold

The following sequence of 418 nucleotides is amplified (chromosome 11, + strand):

1   ttctcacctc agaaaacagg agaatgcaaa catctctcta gctgtatgtg ctctcctgaa
61  ttcgggaggt ggagcaatca aggttaaaat tgaaaatgaa aattatagtc tcactaggga
121 tggcctggga ctagatttgg aagcctctct ttgtaaatgt ctgccctttg tccagtggca
181 cctggacttc acggagagcg aaggctacat ttatatctac gtgaaatcgt ggagccaaga
241 aatctttggg ctgcctattg gcaccctaag aaccaatttg tatgtaagga gcatgtcatc
301 ttctgtacaa gtgagcgccg ctgctgccct ggaatttctc caggacctgg aggaaactgg
361 agggagaccc tgtgtcagac cagagttgcc tgcaagcata gccttccctg aagtggaa

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References

  1. Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A Method and Server for Predicting Damaging Missense Mutations. Nat Methods. 7, 248-249.

  2. Geserick, P., Kaiser, F., Klemm, U., Kaufmann, S. H., and Zerrahn, J. (2004) Modulation of T Cell Development and Activation by Novel Members of the Schlafen (Slfn) Gene Family Harbouring an RNA Helicase-Like Motif. Int Immunol. 16, 1535-1548.

  3. Neumann, B., Zhao, L., Murphy, K., and Gonda, T. J. (2008) Subcellular Localization of the Schlafen Protein Family. Biochem Biophys Res Commun. 370, 62-66.

  4. Schwarz, D. A., Katayama, C. D., and Hedrick, S. M. (1998) Schlafen, a New Family of Growth Regulatory Genes that Affect Thymocyte Development. Immunity. 9, 657-668.

  5. Berger, M., Krebs, P., Crozat, K., Li, X., Croker, B. A., Siggs, O. M., Popkin, D., Du, X., Lawson, B. R., Theofilopoulos, A. N., Xia, Y., Khovananth, K., Moresco, E. M., Satoh, T., Takeuchi, O., Akira, S., and Beutler, B. (2010) An Slfn2 Mutation Causes Lymphoid and Myeloid Immunodeficiency due to Loss of Immune Cell Quiescence. Nat Immunol. 11, 335-343.