Phenotypic Mutation 'dim_sum2' (pdf version)
Alleledim_sum2
Mutation Type nonsense
Chromosome1
Coordinate163,893,230 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Sell
Gene Name selectin, lymphocyte
Synonym(s) CD62L, Ly-22, Lyam1, LECAM-1, Lyam-1, Ly-m22, Lnhr, L-selectin
Chromosomal Location 163,889,556-163,908,354 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a cell surface adhesion molecule that belongs to a family of adhesion/homing receptors. The encoded protein contains a C-type lectin-like domain, a calcium-binding epidermal growth factor-like domain, and two short complement-like repeats. The gene product is required for binding and subsequent rolling of leucocytes on endothelial cells, facilitating their migration into secondary lymphoid organs and inflammation sites. Single-nucleotide polymorphisms in this gene have been associated with various diseases including immunoglobulin A nephropathy. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Oct 2009]
PHENOTYPE: Homozygotes for targeted null mutations exhibit lack of lymphocyte binding to high endothelial venules of peripheral lymph nodes and defects in leukocyte rolling and neutrophil migration into the peritoneum following an inflammatory stimulus. Tumor cellsurvival is also reduced. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_011346, NM_001164059; MGI:98279

MappedYes 
Limits of the Critical Region 164062076 - 164080785 bp
Amino Acid Change Lysine changed to Stop codon
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000027871] [ENSMUSP00000095099] [ENSMUSP00000142237] [ENSMUSP00000141365]
AlphaFold P18337
SMART Domains Protein: ENSMUSP00000027871
Gene: ENSMUSG00000026581
AA Change: K149*

DomainStartEndE-ValueType
CLECT 27 156 1.14e-19 SMART
EGF 159 192 6.55e-1 SMART
CCP 197 254 1.09e-11 SMART
CCP 259 316 1.09e-11 SMART
transmembrane domain 333 355 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000095099
Gene: ENSMUSG00000026581
AA Change: K149*

DomainStartEndE-ValueType
CLECT 27 156 1.14e-19 SMART
CCP 161 218 1.09e-11 SMART
CCP 223 280 1.09e-11 SMART
transmembrane domain 297 319 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000142237
Gene: ENSMUSG00000026581
AA Change: K149*

DomainStartEndE-ValueType
CLECT 27 156 1.14e-19 SMART
CCP 161 218 1.09e-11 SMART
CCP 223 280 1.09e-11 SMART
transmembrane domain 297 319 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000141365
Gene: ENSMUSG00000026581

DomainStartEndE-ValueType
Pfam:Sushi 1 31 1.3e-4 PFAM
transmembrane domain 48 70 N/A INTRINSIC
Predicted Effect probably benign
Meta Mutation Damage Score 0.9755 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(19) : Chemically induced (ENU)(1) Gene trapped(1) Targeted(17)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01981:Sell APN 1 163893195 missense probably benign 0.04
IGL02466:Sell APN 1 163896632 splice site probably null
IGL02578:Sell APN 1 163893165 missense probably damaging 1.00
IGL03243:Sell APN 1 163892911 missense possibly damaging 0.94
dim_sum UTSW 1 163893770 splice site probably null
Duct UTSW 1 163893122 missense probably damaging 1.00
Postit UTSW 1 163893176 missense possibly damaging 0.85
R0125:Sell UTSW 1 163899674 splice site probably benign
R0800:Sell UTSW 1 163893770 splice site probably null
R1900:Sell UTSW 1 163892907 missense probably damaging 1.00
R3848:Sell UTSW 1 163893230 nonsense probably null
R4553:Sell UTSW 1 163899685 missense probably benign 0.08
R4671:Sell UTSW 1 163893042 missense probably damaging 1.00
R4685:Sell UTSW 1 163893829 missense probably damaging 1.00
R4896:Sell UTSW 1 163890631 missense probably benign 0.02
R4970:Sell UTSW 1 163892887 missense possibly damaging 0.75
R5112:Sell UTSW 1 163892887 missense possibly damaging 0.75
R6549:Sell UTSW 1 163893198 missense probably damaging 1.00
R7148:Sell UTSW 1 163893176 missense possibly damaging 0.85
R7545:Sell UTSW 1 163892903 missense probably benign 0.21
R8010:Sell UTSW 1 163893081 missense possibly damaging 0.92
R9026:Sell UTSW 1 163893042 missense probably damaging 1.00
R9239:Sell UTSW 1 163893176 missense possibly damaging 0.85
R9329:Sell UTSW 1 163893122 missense probably damaging 1.00
R9336:Sell UTSW 1 163893177 missense probably damaging 1.00
R9455:Sell UTSW 1 163894218 missense probably benign 0.02
R9699:Sell UTSW 1 163893114 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2016-05-17 10:11 AM by Anne Murray
Record Created 2016-01-07 11:10 PM by Jin Huk Choi
Record Posted 2016-04-27
Phenotypic Description

Figure 1. Homozygous dim_sum2 mice exhibit diminished T-dependent IgG responses to ovalbumin administered with aluminum hydroxide. 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.

Figure 2. Homozygous dim_sum2 mice exhibit diminished frequencies of peripheral blood naïve CD4 T cells in CD4 T cells. CD4 T cell frequency was determined by FACS analysis. 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. Homozygous dim_sum2 mice exhibit diminished frequencies of peripheral blood naïve CD8 T cells in CD8 T cells. CD8 T cell frequency was determined by FACS analysis. 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 dim_sum2 phenotype was identified among G3 mice of the pedigree R3848, some of which showed diminished T-dependent antibody responses to ovalbumin administered with aluminum hydroxide (OVA/Alum) (Figure 1) as well as diminished frequencies of naïve CD4 T cells in CD4 T cells (Figure 2) and naïve CD8 T cells in CD8 T cells (Figure 3).

Nature of Mutation

Figure 4. Linkage mapping of the reduced naïve CD8 T cell frequency in peripheral blood CD8 T cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 68 mutations (X-axis) identified in the G1 male of pedigree R3848. 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 68 mutations. All of the above phenotypes were linked by continuous variable mapping to a mutation in Sell:  an A to T transversion at base pair 164,065,661 (v38) on chromosome 1, or base pair 3,849 in the GenBank genomic region NC_000067 encoding the Sell gene. The strongest linkage was found with a recessive model of inheritance to the reduced naïve CD8 T cell frequency in peripheral blood CD8 T cells, wherein four variant homozygotes departed phenotypically from nine homozygous reference mice and 17 heterozygous mice with a P value of 1.112 x 10-22.   

The mutation corresponds to residue 558 in the mRNA sequence NM_011346 within exon 3 of 9 total exons and residue 558 in the mRNA sequence within exon 3 of 8 total exons.

3834 GATGACGCCTGTCACAAACGAAAGGCAGCTCTC

144  -D--D--A--C--H--K--R--K--A--A--L-

 
Genomic numbering corresponds to NC_000067. The mutated nucleotide is indicated in red.  The mutation results in a substitution of Lys 149 (K149) to a premature stop codon (K149*) in the L-selectin protein, and is strongly predicted by PolyPhen-2 to be probably null (1).
Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 5. Domain structure of L-selectin. The location of the dim_sum2 mutation is shown with a red asterisk. Abbreviations: SP, signal peptide; PRO, pro-peptide; Lectin, C-type lectin domain; EGF, epidermal growth factor-like; SCR, short consensus repeats; TM, transmembrane domain; TAIL, cytoplasmic tail. See the text for more details. This image is interactive; click to view other mutations.

Sell encodes L-selectin (alternatively, CD62L), a lymphocyte-specific member of the selectin family that also includes endothelium-specific E-selectin (CD62E) and platelet-specific P-selectin (CD62P). L-selectin shares similar domains to the other selectins including an extracellular C-type (or calcium-dependent) lectin domain (amino acids 29-156, SMART), an epidermal growth factor (EGF)-like domain (amino acids 159-192), two short consensus repeats (SCRs) (amino acids 197-254 and 259-316), a transmembrane domain (amino acids 333-355), and a cytoplasmic tail (amino acids 356-372) (Figure 5) (2-4). In addition, L-selectin has a signal peptide at amino acids 1-28; SMART) (2;3;5;6).

Alternative splicing of Sell generates two L-selectin isoforms, L-selectin-v1 and L-selectin-v2 (7). The L-selectin-v1 transcript includes a new exon (exon v1) between exon 7 and 8 of Sell. The L-selectin-v2 transcript has an additional 51-bp sequence (exon v2) immediately following the 49-bp L-selectin-v1 insert. L-selectin-v1 and L-selectin-v2 differ from canonical L-selectin at the cytoplasmic tail: L-selectin-v1 has a distinct 24-amino acid sequence (amino acids 362-385) and the eight C-terminal residues of L-selectin-v1 (residues 378–385) are replaced with a distinct 10-amino acid sequence in L-selectin-v2 (residues 378–387) (7).

The dim_sum2 mutation results in substitution of Lys149 for a premature stop codon. Lys149 is within the C-type lectin domain. The expression and localization of the mutant protein has not been examined.

Please see the record dim­_sum for more information about Sell.

Putative Mechanism

L-selectin is a homing receptor that functions in the initial leukocyte tethering to the HEVs and leukocyte rolling on vascular endothelium (8;9). Upon ligand binding, L-selectin clusters and subsequently promotes β1- and β2-integrin activation (10;11) and the mobilization of chemokine receptors (e.g., CXCR4) to the cell surface (12). L-selectin is essential for directing naïve L-selectinhigh lymphocytes to sites of acute and chronic inflammation where naïve T cells can become antigen activated (13-18). Sell-/- mice have impaired T cell responses due to the inability of T cells to migrate into the peripheral lymph nodes (8;9;19-23).

Sell-/- mice exhibited increased humoral immune responses to the T-independent antigen DNP-Ficoll after intraperitoneal (i.p.) injection (24). The antigen-specific IgM response was higher in the Sell-/- mice compared to wild-type mice (24). In addition, the IgG1, IgG3, and IgA responses were also higher in the Sell-/- mice than those in the wild-type mice. After subcutaneous (s.c.) injection, the Sell-/- mice exhibited increased IgM and IgG antibody responses to DNP-Ficoll compared to that in wild-type mice (24). The differences between the Sell-/- and wild-type mice was less significant after s.c. injection when compared to that after i.p. injection. The Sell-/- mice exhibited a lower IgG2b response to DNP-Ficoll than wild-type mice. The T cell proliferative response in draining lymph node cells was reduced five days in the Sell-/- mice after i.p. immunization with Keyhole limpet hemocyanin (KLH), a T-dependent antigen. The amount of anti-KLH IgM, total Ig, IgM, IgG1, IgG2a, and IgE levels were comparable between the Sell-/- and wild-type mice.  Seven days post- s.c. injection of DNP-KLH, the IgM response was reduced in the Sell-/- mice. Taken together, the Sell-/- mice are able to mount humoral immune responses, but they are delayed. The reduced frequency of peripheral blood naïve CD4 T cells mimics that observed in the dim_sum mouse model. Taken together, the phenotype of the dim­_sum2 mice indicates that the mutation leads to aberrant L-selectin function.

Primers PCR Primer
dim_sum2_pcr_F: TTAGTCGCCATACAAAACAAGAGAG
dim_sum2_pcr_R: TTGTGTACCTGCAGATTGCC

Sequencing Primer
dim_sum2_seq_F: AAGCCCTTATTACTACTGGATAGG
dim_sum2_seq_R: CCCAGAGGTGCTATGCTTAAG
Genotyping

Genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transversion.
 

PCR Primers

R38480005_PCR_F: 5’- TTAGTCGCCATACAAAACAAGAGAG-3’

R38480005_PCR_R: 5’- TTGTGTACCTGCAGATTGCC-3’

Sequencing Primers

R38480005_SEQ_F: 5’- AAGCCCTTATTACTACTGGATAGG-3’
 

R38480005_SEQ_R: 5’- CCCAGAGGTGCTATGCTTAAG-3’
 

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 431 nucleotides is amplified (NCBI RefSeq: NC_000067, chromosome 1:164065406-164065836):

ttagtcgcca tacaaaacaa gagagaaatt gagtatttag agaatacatt gcccaaaagc       

ccttattact actggatagg aatcaggaaa attgggaaaa tgtggacatg ggtgggaacc      

aacaaaactc tcactaaaga agcagagaac tggggtgctg gggagcccaa caacaagaag      

tccaaggagg actgtgtgga gatctatatc aagagggaac gagactctgg gaaatggaac      

gatgacgcct gtcacaaacg aaaggcagct ctctgctaca caggtaggca gtgtctaggc      

tcctacgctg atgtgggacc accatagtta gagtaataag actcattacc cagaaactgc      

tattctgggt tctaaaacaa tcctaactaa gccttaagca tagcacctct gggcaatctg      

caggtacaca a

Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (Chr. + strand, A>T).

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsJin Huk Choi, James Butler, Ming Zeng, and Bruce Beutler