Phenotypic Mutation 'Porto' (pdf version)
AllelePorto
Mutation Type missense
Chromosome1
Coordinate58,872,857 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Casp8
Gene Name caspase 8
Synonym(s) MACH, Caspase-8, Mch5, FLICE
Chromosomal Location 58,834,533-58,886,662 bp (+) (GRCm39)
MGI Phenotype FUNCTION: This gene is part of a family of caspases, aspartate-specific cysteine proteases well studied for their involvement in immune and apoptosis signaling. This protein, an initiator of apoptotic cell death, is activated by death-inducing tumor necrosis family receptors and targets downstream effectors. In mouse deficiency of this gene can cause embryonic lethality. This protein may have a role in embryogenesis. Alternative splicing results in multiple transcript variants that encode different protein isoforms. [provided by RefSeq, Apr 2013]
PHENOTYPE: Homozygotes for a targeted null mutation exhibit impaired cardiac muscle development, cardiac erythrocyte congestion, low numbers of colony-forming cells, and prenatal lethality. T-cell restricted knockout mice are viable, but immunodeficient. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_009812 (variant 1), NM_001080126 (variant 2), NM_001277926 (variant 3); MGI: 1261423

MappedYes 
Amino Acid Change Histidine changed to Glutamine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000027189] [ENSMUSP00000127375] [ENSMUSP00000140335] [ENSMUSP00000140546]
AlphaFold O89110
SMART Domains Protein: ENSMUSP00000027189
Gene: ENSMUSG00000026029
AA Change: H244Q

DomainStartEndE-ValueType
DED 1 80 3.21e-23 SMART
DED 99 178 1.01e-15 SMART
CASc 227 480 2.13e-110 SMART
Predicted Effect possibly damaging

PolyPhen 2 Score 0.571 (Sensitivity: 0.88; Specificity: 0.91)
(Using ENSMUST00000027189)
SMART Domains Protein: ENSMUSP00000127375
Gene: ENSMUSG00000026029
AA Change: H244Q

DomainStartEndE-ValueType
DED 1 80 3.21e-23 SMART
DED 99 178 1.01e-15 SMART
CASc 227 480 2.13e-110 SMART
Predicted Effect possibly damaging

PolyPhen 2 Score 0.571 (Sensitivity: 0.88; Specificity: 0.91)
(Using ENSMUST00000165549)
SMART Domains Protein: ENSMUSP00000140335
Gene: ENSMUSG00000026029
AA Change: H264Q

DomainStartEndE-ValueType
DED 21 100 1.5e-25 SMART
DED 119 198 5e-18 SMART
CASc 247 500 1.1e-112 SMART
Predicted Effect possibly damaging

PolyPhen 2 Score 0.894 (Sensitivity: 0.82; Specificity: 0.94)
(Using ENSMUST00000190213)
SMART Domains Protein: ENSMUSP00000140546
Gene: ENSMUSG00000026029
AA Change: H264Q

DomainStartEndE-ValueType
DED 21 100 1.5e-25 SMART
DED 119 198 5e-18 SMART
CASc 247 500 1.1e-112 SMART
Predicted Effect possibly damaging

PolyPhen 2 Score 0.894 (Sensitivity: 0.82; Specificity: 0.94)
(Using ENSMUST00000191201)
Meta Mutation Damage Score 0.1795 question?
Is this an essential gene? Essential (E-score: 1.000) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(17) : Chemically induced (ENU)(2) Chemically induced (other)(1) Endonuclease-mediated(1) Radiation induced(1) Targeted(11) Transgenic(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00684:Casp8 APN 1 58866473 critical splice donor site probably null
IGL00825:Casp8 APN 1 58868165 missense probably benign 0.02
IGL02025:Casp8 APN 1 58863306 missense possibly damaging 0.81
IGL02549:Casp8 APN 1 58872925 missense probably benign
amontillado UTSW 1 58883929 missense probably damaging 1.00
IGL02991:Casp8 UTSW 1 58866438 missense probably benign 0.00
R0609:Casp8 UTSW 1 58883951 missense probably benign 0.00
R0960:Casp8 UTSW 1 58868172 critical splice donor site probably null
R1433:Casp8 UTSW 1 58863283 missense probably damaging 1.00
R1505:Casp8 UTSW 1 58868081 missense probably damaging 0.99
R1506:Casp8 UTSW 1 58863355 missense probably damaging 0.97
R1596:Casp8 UTSW 1 58870833 splice site probably benign
R1674:Casp8 UTSW 1 58883575 missense probably damaging 1.00
R1676:Casp8 UTSW 1 58883575 missense probably damaging 1.00
R1981:Casp8 UTSW 1 58868121 splice site probably null
R3909:Casp8 UTSW 1 58883970 missense probably damaging 1.00
R3911:Casp8 UTSW 1 58872864 missense probably damaging 1.00
R4231:Casp8 UTSW 1 58883929 missense probably damaging 1.00
R4233:Casp8 UTSW 1 58883929 missense probably damaging 1.00
R4234:Casp8 UTSW 1 58883929 missense probably damaging 1.00
R4235:Casp8 UTSW 1 58872857 missense possibly damaging 0.89
R4236:Casp8 UTSW 1 58883929 missense probably damaging 1.00
R4917:Casp8 UTSW 1 58866377 missense probably damaging 1.00
R4918:Casp8 UTSW 1 58866377 missense probably damaging 1.00
R5063:Casp8 UTSW 1 58883533 missense probably damaging 1.00
R5092:Casp8 UTSW 1 58883835 missense possibly damaging 0.53
R5153:Casp8 UTSW 1 58884004 missense probably benign 0.00
R5964:Casp8 UTSW 1 58872895 missense possibly damaging 0.62
R5979:Casp8 UTSW 1 58868071 missense probably benign
R7602:Casp8 UTSW 1 58872898 missense probably benign 0.43
R7675:Casp8 UTSW 1 58863106 missense possibly damaging 0.69
R8272:Casp8 UTSW 1 58872901 missense probably damaging 0.96
R8714:Casp8 UTSW 1 58872812 missense possibly damaging 0.57
R8747:Casp8 UTSW 1 58883617 missense probably benign 0.00
R9279:Casp8 UTSW 1 58883542 missense probably benign 0.20
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:31 PM by Anne Murray
Record Created 2019-01-22 10:45 AM by Bruce Beutler
Record Posted 2019-02-14
Phenotypic Description

Figure 1. Porto mice exhibit increased frequencies of peripheral effector memory CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 Porto phenotype was identified among G3 mice of the pedigree R4235, some of which showed increased frequencies of effector memory CD8 T cells in CD8 T cells in the peripheral blood (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the increased effector memory CD8 T cell frequency using a dominant model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 62 mutations (X-axis) identified in the G1 male of pedigree R4235. 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 from pedigree R4235 identified 62 mutations. The effector memory CD8 T cell phenotype was linked by continuous variable mapping to mutations in two genes on chromosome 1: Tyw5 and Casp8. The mutation in Casp8 was presumed causative as the phenotype in Porto mimics that of other Casp8 mutant alleles (see MGI). The mutation in Casp8 is a T to A transversion at base pair 58,833,698 (GRCm38) on chromosome 1, or base pair 38,471 in the GenBank genomic region NC_000067 encoding caspase-8. Linkage was found with a dominant model of inheritance, wherein one homozygous variant mouse and nine heterozygous mice departed phenotypically from 10 homozygous reference mice with a P value of 0.000407 (Figure 2).

The mutation corresponds to residue 1,125 in the mRNA sequence NM_009812, within 7 of 9 exons.

1108 CTGATCATCAACAATCATGATTTCAGCAAGGCC
239  -L--I--I--N--N--H--D--F--S--K--A-

The mutated nucleotide is indicated in red.  The mutation results in a histidine to glutamine substitution at position 244 (H244Q) in the caspase-8 protein (isoform 1), and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.571).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain organization of mouse caspase-8.  The protease domain consists of two subunits (larged, p20; small, p10). Abbreviations: DED, death effector domain.  The Porto mutation results in a histidine to glutamine substitution at position 244. This image is interactive. Other mutations found in caspase-8 are noted in red. Click on each allele for more information.

Caspase-8 is one of 14 caspase proteins (cysteine-dependent aspartate-specific proteases) found in mice. These enzymes cleave protein substrates after Asp residues to propagate signaling leading to apoptosis (caspase-2, -3, -6, -7, -8, -9, -10) or inflammation (caspase-1, -4, -5, -8); capsases may also have roles in other processes such as differentiation (caspase-14) (1). The apoptotic caspases are classified as initiator (caspase-2, -8, -9, -10) or effector caspases (caspase-3, -6, -7), the effector caspases being activated by initiator caspases, whereas initiator caspases are activated by non-caspases through a mechanism involving clustering leading to dimerization. 

Caspases are initially produced as zymogens (procaspases). The structural organization of all procaspases is similar, consisting of an N-terminal prodomain and a C-terminal catalytic domain (Figure 3) (2). In caspase-8, two tandem death effector domains (DEDs) contained within the prodomain mediate clustering at death receptor-adapter complexes to facilitate caspase dimerization critical for catalytic activation (3). The caspase catalytic domain is composed of two subunits (large, p20; and small, p10) covalently connected by the intersubunit linker. Autoproteolytic cleavage of the linker in caspase-8 occurs following dimerization, separating the large and small subunits of the catalytic domain; subsequently the prodomain is cleaved from the large subunit (4)

The Porto mutation results in a histidine to glutamine substitution at position 244 (H244Q); amino acid 244 is within an undefined region between the DED2 and caspase p20 domains in the prodomain.

Please see the record amontillado for more information about Casp8.

Putative Mechanism

Caspases are critical components of the signaling pathways leading to apoptosis, or programmed cell death. Ligand binding to death receptors results in receptor aggregation at the cell membrane and the subsequent clustering of downstream signaling complexes (5;6).  In response to activation by Fas ligand (FasL; see the record for riogrande), the adapter FADD is recruited to the receptor via DD-DD interactions; the DED of FADD then recruits caspase-8 or caspase-10 via DED-DED interactions. The ternary complex (containing Fas, FADD, and caspase-8/10) is called the death-inducing signaling complex (DISC) (6-10), and activates caspase-8 by proximity-induced dimerization. Caspase-8 cleaves effector caspases (caspase-3 and caspase-7) that go on to degrade cellular organelles during the process of apoptosis.  Caspase-8 also participates in proapoptotic signaling induced by TRAIL receptors 1 and 2 [reviewed in (11)] and by TNFR1 (12;13)

Although best known as an initiator caspase necessary for apoptosis triggered by death receptor activation, caspase-8 is also involved in IL-1 receptor-dependent inflammatory signaling by virtue of its role in the proteolytic processing of IL-1β to its mature form [reviewed in (14)]. In addition to directly processing pro-IL-1β, caspase-8 serves as an initiator caspase for the activation of caspase-1, which directly processes pro-IL-1β, in inflammasomes (15-18)

Caspase-8 deficiency in mice results in embryonic lethality at E10.5 (19). T cell-specific caspase-8 deficiency in mice (tcasp8-/-) resulted in defective T cell proliferation and homeostasis despite normal thymocyte development, leading to reduced peripheral T cells (20). The reduction in T cells is thought to stem from increased necroptosis upon activation because RIPK3 knockout restored normal T cell frequencies (21). tcasp8-/- T cells displayed impaired responses to activation stimuli, and showed activation marker upregulation and stimulation-independent proliferation of peripheral T cells (20;22)tcasp8-/- mice failed to mount a T cell-mediated immune response to LCMV infection (20). Moreover, they developed an age-dependent lethal lymphoproliferative disorder different from autoimmune lymphoproliferative syndrome (ALPS) characterized by lymphoadenopathy, splenomegaly, and accumulation of T cell infiltrates in the lungs, liver, and kidneys (22). These findings are consistent with a report of caspase-8 deficiency in humans caused by a point mutation (R248W) in the large subunit of the catalytic domain (23). Homozygous individuals exhibited lymphoadenopathy and splenomegaly, along with defective activation of T cells, B cells, and NK cells that resulted in immunodeficiency (23).

The Porto mutation may impair the interaction of caspase-8 with its substrates. However, unlike mice with a null mutation of Casp8 (19), homozygous Porto mice are born alive and survive past weaning age, suggesting that the mutant caspase-8 protein produced in Porto mice retains some function. The level of function, while sufficient to support embryonic development, was inadequate for normal T cell proliferation and homeostasis.

Primers PCR Primer
Porto_pcr_F: GCAATGGAAGGAGCCTATGC
Porto_pcr_R: CCAGTATAGGGTAATGCCAGG

Sequencing Primer
Porto_seq_F: GCCTATGCTTGCCTGAGG
Porto_seq_R: GGATTTTCTGGATAGCATTTGAAATG
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 561 nucleotides is amplified (chromosome 1, + strand):


1   gcaatggaag gagcctatgc ttgcctgagg ggctgagcag caatccagct tcttcctgag
61  cacttggaca tagccagtgt ctgagctgca gcatcaaccc ctggattggg cttgtgtttt
121 ccagacttca gacaaagttt accaaatgaa gaacaaacct cggggatact gtctgatcat
181 caacaatcat gatttcagca aggcccggga agacataacc caactccgaa aaatgaagga
241 cagaaaagga acagactgtg ataaaggtat ggtgggatcc aaaggagaag catgagaaaa
301 ttttattctt atggaccttt ctttaaccaa aaaggagaga gcagtagtta tacttaaagg
361 gatatatata tatatatata tatatatata tataaattaa ttaaagcatt tattatgttg
421 aaaattttta aacaattttt attagatatt ttcttcattt acatttcaaa tgctatccag
481 aaaatcccca atacccttcc cccgccctgc tccccaaccc acccattccc acttcctgcc
541 cctggcatta ccctatactg g


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

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsXue Zhong, Jin Huk Choi, and Bruce Beutler