Phenotypic Mutation 'arrested' (pdf version)
Allelearrested
Mutation Type missense
Chromosome8
Coordinate10,081,545 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Tnfsf13b
Gene Name tumor necrosis factor (ligand) superfamily, member 13b
Synonym(s) BLyS, TALL-1, zTNF4, D8Ertd387e, BAFF
Chromosomal Location 10,056,229-10,086,000 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFFR. This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator. It has been also shown to play an important role in the proliferation and differentiation of B cells. Alternatively spliced transcript variants encoding distinct isoforms have been identified. [provided by RefSeq, Mar 2011]
PHENOTYPE: Homozygous null mice have reduced number of B cells and reduced levels of immunoglobulins. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_033622, NM_001347309; MGI:1344376

MappedYes 
Amino Acid Change Isoleucine changed to Valine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000033892] [ENSMUSP00000146694] [ENSMUSP00000146904]
AlphaFold Q9WU72
SMART Domains Protein: ENSMUSP00000033892
Gene: ENSMUSG00000031497
AA Change: I217V

DomainStartEndE-ValueType
low complexity region 48 67 N/A INTRINSIC
TNF 169 308 1.88e-2 SMART
Predicted Effect probably benign

PolyPhen 2 Score 0.016 (Sensitivity: 0.95; Specificity: 0.79)
(Using ENSMUST00000033892)
Predicted Effect possibly damaging

PolyPhen 2 Score 0.480 (Sensitivity: 0.89; Specificity: 0.90)
(Using ENSMUST00000207792)
Predicted Effect probably benign
Meta Mutation Damage Score 0.1596 question?
Is this an essential gene? Probably nonessential (E-score: 0.081) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(11) : Chemically induced (ENU)(3) Endonuclease-mediated(1) Targeted(5) Transgenic(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01016:Tnfsf13b APN 8 10081612 missense probably damaging 1.00
IGL01383:Tnfsf13b APN 8 10081528 missense probably damaging 0.98
IGL01650:Tnfsf13b APN 8 10081411 missense probably damaging 1.00
Applecrisp UTSW 8 10081534 missense probably damaging 1.00
Frozen UTSW 8 10081661 splice site probably null
F5493:Tnfsf13b UTSW 8 10056916 missense probably damaging 1.00
R0610:Tnfsf13b UTSW 8 10081661 splice site probably null
R0723:Tnfsf13b UTSW 8 10057166 splice site probably null
R1435:Tnfsf13b UTSW 8 10085358 missense probably benign 0.06
R1648:Tnfsf13b UTSW 8 10081534 missense probably damaging 1.00
R1744:Tnfsf13b UTSW 8 10081661 splice site probably null
R2266:Tnfsf13b UTSW 8 10057306 missense probably benign 0.23
R3723:Tnfsf13b UTSW 8 10081545 missense possibly damaging 0.48
R5230:Tnfsf13b UTSW 8 10081608 missense possibly damaging 0.80
R5913:Tnfsf13b UTSW 8 10056988 missense probably damaging 1.00
R6741:Tnfsf13b UTSW 8 10057314 missense possibly damaging 0.66
R7310:Tnfsf13b UTSW 8 10081651 nonsense probably null
R7882:Tnfsf13b UTSW 8 10057078 missense not run
R8420:Tnfsf13b UTSW 8 10056795 start gained probably benign
R9124:Tnfsf13b UTSW 8 10056966 missense probably benign
R9374:Tnfsf13b UTSW 8 10085391 missense possibly damaging 0.74
R9474:Tnfsf13b UTSW 8 10081648 missense probably damaging 1.00
Z1177:Tnfsf13b UTSW 8 10085427 missense probably benign 0.26
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:30 PM by Anne Murray
Record Created 2019-01-23 10:42 AM by Bruce Beutler
Record Posted 2019-02-14
Phenotypic Description

Figure 1. Arrested mice exhibit reduced B to T ratios. Flow cytometric analysis of peripheral blood was utilized to determine B and T cell frequencies. 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.

Figure 2. Arrested mice exhibit decreased frequencies of peripheral B cells. Flow cytometric analysis of peripheral blood was utilized to determine B 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.

Figure 3. Arrested mice exhibit decreased frequencies of peripheral IgM+ B cells. Flow cytometric analysis of peripheral blood was utilized to determine B 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 arrested phenotype was identified among N-nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R3723, some of which showed a reduced B:T cell ratio (Figure 1) due to reduced frequencies of B cells (Figure 2) and IgM+ B cells (Figure 3) in the peripheral blood. 

Nature of Mutation

Figure 4. Linkage mapping of the reduced B cell frequency using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 41 mutations (X-axis) identified in the G1 male of pedigree R3723. 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 41 mutations. The above anomalies were linked by continuous variable mapping to a mutation in Tnfsf13b:  an A to G transition at base pair 10,031,545 (v38) on chromosome 8, or base pair 25,343 in the GenBank genomic region NC_000074 encoding Tnfsf13b. The strongest association was found with a recessive model of linkage to the normalized B cell frequency phenotype, wherein 10 variant homozygotes departed phenotypically from 18 homozygous reference mice and 23 heterozygous mice with a P value of 7.014 x 10-5 (Figure 4).  

The mutation corresponds to residue 916 in the mRNA sequence NM_033622 within exon 6 of 7 total exons and residue 1,025 in the mRNA sequence NM_001347309 within exon 5 out of 6 total exons.

25328 GCTATGGGTCATGTCATCCAGAGGAAGAAAGTA

231   -A--M--G--H--V--I--Q--R--K--K--V- (isoform 1; NP_296371)

212   -A--M--G--H--V--I--Q--R--K--K--V- (isoform 2; NP_001334238)

Genomic numbering corresponds to NC_000074. The mutated nucleotide is indicated in red. The mutation results in an isoleucine to valine substitution at position 236 (I236V) in isoform 1 of the BAFF protein (PolyPhen score = 0.480) and an isoleucine to valine substitution at position 217 in isoform 2 of the BAFF protein (PolyPhen score = 0.016).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 5. Domain structure of BAFF. The arrested mutation results in an isoleucine to valine substitution at position 236. This image is interactive. Click on each alleleAbbreviations: CYTO, cytoplasmic domain; TM, transmembrane domain; ECD, extracellular domain; THD, TNF homology domain. TNF is cleaved by furin-like proteases to produce soluble BAFF. See text for more details. Image is interactive; click to view information about other BAFF mutations.

Tnfsf13b encodes B cell activating factor (BAFF; alternatively, BLyS, TALL-1, zTNF-4, THANK, or TNFSF13), a member of the tumor necrosis factor (TNF) family. BAFF is a type II transmembrane protein that undergoes cleavage between Arg126 and Ala127 by furin-like proteases to generate a soluble form of BAFF (1-3). BAFF contains a single transmembrane domain at amino acids 48-68 and a TNF homology domain (THD) at amino acids 169-308 (Figure 5).

Mouse Tnfsf13b can undergo alternative splicing that results in skipping of exon 4 (exon 4 encodes the first β-sheet of the THD (amino acids 156-184); Gly185 is substituted with an Arg) and creation of a functional N-linked glycosylation site at Asn155 to produce a splice variant called ΔBAFF [(4); reviewed in (5)]. The ΔBAFF isoform is able to assemble into disulfide-linked complexes with itself as well as full-length BAFF (4). Association of ΔBAFF with BAFF prevents release of sBAFF and results in formation of inactive homotrimers (4;6).

The arrested mutation (I217V) is within the THD. Alteration of the THD may result in aberrant association of BAFFarrested with its receptors BCMA, TACI (transmembrane activator and calcium modulator ligand (CAML) interactor), and BAFFR.

Please see the record Frozen for more information about Tnfsf13b.

Putative Mechanism

BAFF/BAFFR activates the alternative NF-κB (NF-κB2) signaling pathway (see the record for xander) to mediate the survival and maturation of splenic B cells (7;8). BAFF/BAFFR-induced NF-κB2 signaling promotes B cell survival by upregulating integrins that retains autoreactive B cells in the splenic marginal zone (9). In addition, ERK activation is sustained and there is an increased turnover of Bim, a proapoptotic protein (7;8;10). BAFF activates the classical NF-κB (NF-κB1) signaling pathway (see the record for Finlay) to regulate immunoglobulin class switching through an induction of activation induced deaminase (AID) and to generate antibodies (9). BAFF also induces the protein kinase Cδ (PKCδ)-mediated nuclear signaling pathway to regulate B-cell survival (11). BAFF stimulates the Akt/mTOR signaling pathway to promote cell growth and survival (10;12;13). BAFF is required for the maintenance, but not initiation, of the germinal center (GC) reaction (14).  Elevated circulating levels of BAFF have been observed in patients with rheumatoid arthritis, multiple sclerosis, SLE, and SS. Total numbers of cells in the thymus, bone marrow, and PEC were comparable between the Tnfsf13b-/- and wild-type mice (15;16). The number of B cells in the spleen and lymph nodes were reduced in Tnfsf13b-/- mice compared to wild-type mice (15;17). The percentage of peripheral B1 B cells in the Tnfsf13b-/- mice was comparable to that in wild-type mice; however, the B2 (B220+, CD23hi) B cell percentage in the periphery was reduced compared to wild-type mice (15;16). In contrast, inhibition of BAFF results in reduced splenic B1a and B1b cells, but peritoneal B1 cells are unaffected (18). T-dependent and T-independent humoral responses were diminished in BAFF-deficient (Tnfsf13b-/-) mice (15-17;19-21). The phenotype of the arrested mice indicates loss of BAFF-associated function.

Primers PCR Primer
arrested_pcr_F: GGCTTCTCAGCTTTAAAAGAGG
arrested_pcr_R: TCCCAAAGCAGCACTTTTGG

Sequencing Primer
arrested_seq_F: CTCAGCTTTAAAAGAGGAAATGCC
arrested_seq_R: AGGACAGCTACATACCAG
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 412 nucleotides is amplified (chromosome 8, + strand):


1   ggcttctcag ctttaaaaga ggaaatgcct tggaggagaa agagaacaaa atagtggtga
61  ggcaaacagg ctatttcttc atctacagcc aggtagcgtc aaactctccc ctacccctgg
121 tccgacgttt tcactgtcct tcacgtctgg tgacatatcc cggcttgttt ctcaggttct
181 atacacggac cccatctttg ctatgggtca tgtcatccag aggaagaaag tacacgtctt
241 tggggacgag ctgagcctgg tgaccctgtt ccgatgtatt cagaatatgc ccaaaacact
301 gcccaacaat tcctgctact cggctggtat gtagctgtcc taagacctgc ttagcaagat
361 ggtgtagatt gtgctgggga atcttattcc ccccaaaagt gctgctttgg ga


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