Phenotypic Mutation 'secro' (pdf version)
Allelesecro
Mutation Type intron
Chromosome8
Coordinate111,315,567 bp (GRCm38)
Base Change A ⇒ G (forward strand)
Gene Mlkl
Gene Name mixed lineage kinase domain-like
Synonym(s) 9130019I15Rik
Chromosomal Location 111,311,797-111,338,177 bp (-)
MGI Phenotype FUNCTION: This gene belongs to the protein kinase superfamily. The encoded protein contains a protein kinase-like domain; however, is thought to lack protein kinase activity. This protein plays a critical role in tumor necrosis factor (TNF)-induced necroptosis, a programmed cell death process, via interaction with receptor-interacting protein 3 (Rip3), which is a key signaling molecule in necroptosis pathway. Knockout of this gene in mice showed that it is essential for necroptosis. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2015]
PHENOTYPE: Mice homozygous for a knock-out allele exhibit imapired macrophage and mouse embryonic fibroblast necroptosis. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001310613 (isoform 1) NM_029005 (isoform 2); MGI:1921818

Mapped Yes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000055521] [ENSMUSP00000113718]
PDB Structure Structure of MLKL [X-RAY DIFFRACTION]
Crystal structure of the mouse MLKL kinase-like domain [X-RAY DIFFRACTION]
Crystal structure of the mouse RIP3-MLKL complex [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000055521
Gene: ENSMUSG00000012519

DomainStartEndE-ValueType
low complexity region 109 115 N/A INTRINSIC
Pfam:Pkinase_Tyr 195 448 2.7e-41 PFAM
Pfam:Pkinase 200 450 2.1e-30 PFAM
Pfam:Kinase-like 270 438 1.6e-7 PFAM
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000113718
Gene: ENSMUSG00000012519

DomainStartEndE-ValueType
low complexity region 109 115 N/A INTRINSIC
Pfam:Pkinase_Tyr 195 453 3.3e-42 PFAM
Pfam:Pkinase 196 453 1.4e-33 PFAM
Pfam:Kinase-like 270 438 8.9e-8 PFAM
Predicted Effect probably benign
Meta Mutation Damage Score 0.0898 question?
Is this an essential gene? Probably nonessential (E-score: 0.074) question?
Phenotypic Category
Phenotypequestion? Literature verified References
LPS-induced Necroptosis - decreased 22265413
Macrophage necroptosis: low 22265413
TLR signaling defect: hypersensitivity to CpG + IFNg
Candidate Explorer Status CE: excellent candidate; Verification probability: 0.587; ML prob: 0.558; human score: 3.5
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(4) : Chemically induced (ENU)(1) Endonuclease-mediated(1) Gene trapped(1) Targeted(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00089:Mlkl APN 8 111319428 nonsense probably null
IGL01376:Mlkl APN 8 111319747 missense probably damaging 1.00
IGL02801:Mlkl APN 8 111316432 missense probably benign 0.18
IGL02965:Mlkl APN 8 111331837 missense probably benign 0.31
IGL03121:Mlkl APN 8 111314980 missense probably damaging 1.00
Ghoulish UTSW 8 111322748 missense probably damaging 1.00
mecro UTSW 8 111319716 critical splice donor site probably null
necro UTSW 8 111312100 intron probably benign
R0133:Mlkl UTSW 8 111327948 missense probably damaging 1.00
R0230:Mlkl UTSW 8 111315062 missense probably benign 0.07
R0387:Mlkl UTSW 8 111333350 missense probably damaging 1.00
R0497:Mlkl UTSW 8 111327873 missense probably damaging 1.00
R0735:Mlkl UTSW 8 111327801 unclassified probably benign
R1733:Mlkl UTSW 8 111322748 missense probably damaging 1.00
R1761:Mlkl UTSW 8 111333723 missense possibly damaging 0.81
R1911:Mlkl UTSW 8 111312100 intron probably benign
R2057:Mlkl UTSW 8 111333610 missense probably benign 0.07
R2921:Mlkl UTSW 8 111316447 missense probably benign 0.02
R3745:Mlkl UTSW 8 111315567 intron probably benign
R4760:Mlkl UTSW 8 111319716 critical splice donor site probably null
R5377:Mlkl UTSW 8 111327937 missense probably benign 0.23
R7052:Mlkl UTSW 8 111319442 missense possibly damaging 0.65
R7155:Mlkl UTSW 8 111319403 missense probably damaging 1.00
R7459:Mlkl UTSW 8 111333530 missense probably benign 0.36
R7728:Mlkl UTSW 8 111333619 missense probably damaging 1.00
R8036:Mlkl UTSW 8 111333454 missense probably damaging 1.00
R8064:Mlkl UTSW 8 111312068 missense probably benign 0.38
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:44 PM by Diantha La Vine
Record Created 2015-12-01 1:04 AM by Ying Wang
Record Posted 2015-12-21
Phenotypic Description

Figure 1. Secro mice exhibited resistance to necroptosis in response to TLR4 ligand, LPS. 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 secro phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R3745, some of which exhibited resistance to lipopolysaccharide (LPS)-induced necroptosis (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the resistance to LPS-induced necroptosis 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 R3745. 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 necroptosis phenotype was linked by continuous variable mapping to a mutation in Mlkl: a T to C transition at base pair 111,315,567 (v38) on chromosome 8, or base pair 22,890 in the GenBank genomic region NC_000074. Linkage was found with a recessive model of inheritance, wherein five variant homozygotes departed phenotypically from nine homozygous reference mice and 13 heterozygous mice with a P value of 1.245 x 10-20 (Figure 2). A substantial semidominant effect was observed, but the mutation is preponderantly recessive. 

 

The secro mutation is within intron eight, 25 base pairs away from exon 9 (out of 11 total exons).

 

             <--exon 8                   <--intron 8-->                     exon 9-->           <--exon 11
22074 ……GCTGAAATATATAG gtatgttcctgtacagattctaact……tgacaagcccttggttttcttttag CTTTGGAATTGTA…… ……AAGAAGGTGTAA
380   ……-A--E--I--Y--S                                                      --F--G--I--V-…… ……-K--K--V--*- 472 (isoform 1; NP_083281)
380   ……-A--E--I--Y--S                                                      --F--G--I--V-…… ……-K--K--V--*- 464 (isoform 2; NP_001297542)

 

Genomic numbering corresponds to NC_000074. The mutated nucleotide is indicated in red, the splice donor site is in green, and the splice acceptor site is in blue. 

 

 

The effect of the secro mutation on the mRNA sequence and MLKLsecro protein expression is unknown. In the event of exon 9 skipping, the aberrant transcript would have a deletion of the 50 base pair exon 9, leading to a frame-shift occurring after amino acid 383 of the protein (isoform 1), and termination after the inclusion of two aberrant amino acids.

 

             <--exon 8                   <--intron 8-->                     exon 9-->       exon 10-->
22074 ……GCTGAAATATATAG gtatgttcctgtacagattctaact……tgacaagcccttggttttcttttag CTTTGGAATTGTA…… GCTGTGA…… 

 
380   ……-A--E--I--Y--R                                                      --F--G--I--V-…… --L--*- (isoform 1; NP_083281)

           correct                                                             deleted      aberrant

 

Genomic numbering corresponds to NC_000074. The mutated nucleotide is indicated in red, the splice donor site is in green, the splice acceptor site is in blue, the aberrant amino acid in exon 8 is denoted by italics. 

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain organization of MLKL. MLKL has an N-terminal four-helical bundle (amino acids 1-130) followed by a two-helix linker (termed brace; amino acids 131-170) that tethers the N-terminus to a pseudokinase domain (amino acids 171-464). The secro mutation is within intron 8. This image is interactive. Other MLKL mutations are noted in red. Click on each mutation for more information.

Mlkl encodes mixed lineage kinase domain-like (MLKL), a pseudokinase and member of the protein kinase superfamily. MLKL has a pseudokinase domain (amino acids 171-464), but does not exhibit kinase activity (1) (Figure 3). Similar to kinases, MLKL has a (Val-Ala-Ile-Lys) motif, which positions the α- and β-phosphates of ATP during phosphoryl transfer, but does not have a catalytic loop or an activation loop. The pseudokinase domain has a kinase fold with N- and C-lobes (1-3). The N-lobe has an antiparallel, five-stranded β-sheet and an α-helix (helix αC), whereas the C-lobe contains seven α-helices and a pair of β-strands (4). MLKL has an N-terminal four-helical bundle (amino acids 1-130) followed by a two-helix linker (termed brace; amino acids 131-170) that tethers the N-terminus to a pseudokinase domain (1).

 

For more information about Mlkl, please see the record for necro.

Putative Mechanism

Necroptosis is a pro-inflammatory form of cell death regulated by the kinases RIP1 and RIP3. Necroptosis occurs after stimulation of the DNA receptor, DNA-dependent activator of interferon regulatory factors (DAI), or activation of death receptors [e.g., TNF receptor 1 (TNFR1; see the record PanR1 for information about TNF) and Fas (see the record for cherry)], Toll-like receptors [TLRs; e.g., TLR3 and TLR4 (see the record for lps3)], T-cell antigen receptor (TCR), or interferon receptor [IFNAR1 (see the record for macro-1) and IFNAR2 (see the record for macro-2)] signaling. During necroptosis, RIP3 binds RIP1 through their respective RIP homotypic interaction motif domains, forming the necroptosome. RIP1 and RIP3 phosphorylation in the necroptosome leads to the recruitment of MLKL (5;6). MLKL is essential for necroptosis (2;4-6). Inhibition of MLKL function using necrosulfonamide prevents necrosome formation and subsequent necropototic signaling (5;6). Mouse dermal fibroblasts (MDFs), mouse embryonic fibroblasts (MEFs), and bone-marrow-derived macrophages (BMDMs) derived from the Mlkl-/- mice were resistant to TNF-induced necroptotic cell death (1).  

 

The secro mice exhibit resistance to LPS-induced necroptosis; necroptosis downstream of TNF has not been examined in the secro mice. The phenotype of the secro mice indicates loss of MLKLsecro function.

Primers PCR Primer
secro_pcr_F: TACATTAGAGAACCAAGAGTCCAGG
secro_pcr_R: AATCATTGCAGGGACACGGG

Sequencing Primer
secro_seq_F: GGACAACACAGATAGGAAATTCC
secro_seq_R: CTGCTTGGAACTGGAATTACAG
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 753 nucleotides is amplified (chromosome 8, - strand):


1   aatcattgca gggacacggg aaaagaaatg tgctgagtgc ttggtttctt ggttttattt
61  tgaaacctta aagatatact tattttttat tttatgtgta tgagtgttat gtcaacatgt
121 atatacacca tgtgcagtgt caacagaggc cagaagaggg catctgatct gcttggaact
181 ggaattacag atggttgtga attgccatgt gggtgctagg aactgaaccc aggttagcaa
241 atactctaac cattgagcca tctctccagc ccatttaaaa aaaattttta atgtatctgt
301 gtgtgtttgt gtgtgtgtgt gtgagagaga gagagagaga gagagagaga gagagagaga
361 gggagaggga gagggagagg gagtgagaga gagagagaga gagagagaga gagagagggg
421 tggggggtgt acataacaca gaatgtgtgt ggatgccaga gatcaacctt cttttagggc
481 catttctctc cttccacctt tatgtaattc tggagattaa atttaaaaga gtgtggcaca
541 gtgcaagctc tttgcctgcc aagtcaccca cctgacaagc ccttggtttt cttttagctt
601 tggaattgta ctctgggaaa ttgccactgg aaagatccca tttgaaggtg aagaacatga
661 tggctggctc tactggggtt ggggttaggt cctcttctga gaggacagga atttcctatc
721 tgtgttgtcc tggactcttg gttctctaat gta


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

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsYing Wang, Hexin Shi, Zhao Zhang, Lei Sun, Doan Dao, and Bruce Beutler