Phenotypic Mutation 'blossom'

• Allele: blossom
• Mutation Type: missense
• Chromosome: 16
• Coordinate: 19,775,889 bp (GRCm39)
• Base Change: T ⇒ A (forward strand)
• Gene: Klhl6
• Gene Name: kelch-like 6
• Chromosomal Location: 19,765,242-19,801,766 bp (-) (GRCm39)
• MGI Phenotype: FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a member of the kelch-like (KLHL) family of proteins, which is involved in B-lymphocyte antigen receptor signaling and germinal-center B-cell maturation. The encoded protein contains an N-terminal broad-complex, tramtrack and bric a brac (BTB) domain that facilitates protein binding and dimerization, a BTB and C-terminal kelch (BACK) domain, and six C-terminal kelch repeat domains. Naturally occurring mutations in this gene are associated with chronic lymphocytic leukemia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2017] ; PHENOTYPE: Mice homozygous for a knock-out allele exhibit spleen hypoplasia, defects in mature B-cell subsets with normal pro- and pre-B-cell development, severely impaired antigen-dependent germinal center formation, and reduced memory IgG response. [provided by MGI curators]
• Mapped: Yes
• Limits of the Critical Region: 19946499 - 19983037 bp
• Amino Acid Change: Aspartic acid changed to Valine
• Institutional Source: Beutler Lab
• Gene Model: predicted gene model for protein(s): [ENSMUSP00000053023] [ENSMUSP00000130755]
• AlphaFold: Q6V595
• SMART Domains: Protein: ENSMUSP00000053023; Gene: ENSMUSG00000043008; AA Change: D223V

Domain	Start	End	E-Value	Type
BTB	70	167	1.43e-25	SMART
BACK	172	274	1.68e-35	SMART
Kelch	376	419	3.05e-1	SMART
Kelch	420	466	6.82e-11	SMART
Kelch	467	514	4.27e-3	SMART
Kelch	515	556	3.06e-4	SMART
Kelch	557	604	3.47e-3	SMART

• Predicted Effect: probably damaging ; PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
  (Using ENSMUST00000058839)
• SMART Domains: Protein: ENSMUSP00000130755; Gene: ENSMUSG00000043008

Domain	Start	End	E-Value	Type
Pfam:BTB	60	98	1.2e-9	PFAM

• Predicted Effect: noncoding transcript
• Meta Mutation Damage Score: 0.9688
• Is this an essential gene?: Probably nonessential (E-score: 0.086)
• Phenotypic Category: Autosomal Recessive
• Mode of Inheritance: Autosomal Recessive
• Last Updated: 2019-09-04 9:41 PM by Diantha La Vine
• Record Created: 2016-12-07 10:31 PM by Jin Huk Choi
• Record Posted: 2018-07-20
• Science Writers: Anne Murray
• Illustrators: Diantha La Vine
• Authors: Xue Zhong, Bruce Beutler

Accession Number

NCBI RefSeq: NM_183390; MGI:2686922

Alleles Listed at MGI

All Mutations and Alleles(5) : Chemically induced (ENU)(1) Targeted(4)

Phenotypic Description

Figure 1. Blossom mice exhibit a reduced B to T cell ratio. Flow cytometric analysis of peripheral blood was utilized to determine B and 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.

Figure 2. Blossom mice exhibit increased frequencies of peripheral 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.

Figure 3. Blossom mice exhibit increased frequencies of peripheral CD4+ 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.

Figure 4. Blossom mice exhibit increased frequencies of peripheral 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 blossom phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4833, some of which showed a decrease in the B to T cell ratio (Figure 1) caused by a increased frequencies of total T cells (Figure 2), CD4+ T cells (Figure 3), and CD8+ T cells (Figure 4), all in the peripheral blood. 

Nature of Mutation

Figure 5. Linkage mapping of the increased frequency of T cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 71 mutations (X-axis) identified in the G1 male of pedigree R4833. 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.

hole exome HiSeq sequencing of the G1 grandsire identified 71 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Klhl6:  an A to T transversion at base pair 19,957,139 (v38) on chromosome 16, or base pair 25,911 in the GenBank genomic region NC_000082 encoding Klhl6. The strongest association was found with a recessive model of linkage to the normalized frequency of total T cells, wherein four variant homozygotes departed phenotypically from 11 homozygous reference mice and 16 heterozygous mice with a P value of 1.451 x 10^-7 (Figure 5).  A substantial semidominant effect was observed in most of the assays. 

The mutation corresponds to residue 714 in the mRNA sequence NM_183390 within exon 3 of 7 total exons.

698 CACATCTTGAGGAGTGACGACCTGTGTGTGACT 218 -H--I--L--R--S--D--D--L--C--V--T-

The mutated nucleotide is indicated in red.  The mutation results in an aspartic acid (D) to valine (V) substitution at position 233 (D223V) in the KLHL6 protein (Figure 6), and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Protein Prediction

Figure 6. Domain structure of the KLHL6 protein. KLHL6 is a member of the Kelch-like family. The blossom mutation (D223V) is indicated. Please see the text for more details about these domains. BTB, Broad-complex, Tramtrack and Bric à brac domain; BACK, BTB and C-terminal kelch domain. The image is interactive. Additional mutations found in KLHL6 are noted. Click each mutation to view more information. Domain information is from SMART and UniProt.

KLHL6 is a member of the Kelch-like family (see the record for teeny for information about KBTBD2, another Kelch-like family member). Similar to other Kelch-like family members, KLHL6 has a Bric-a-brac, Tramtrack, Broad-complex (BTB) domain at the N-terminus, a BACK domain, and a kelch repeat region at the C-terminus (KLHL6 has five or six kelch repeats) (Figure 6) (1). BTB domains facilitate protein-protein interactions, the BACK domain has no known function, and Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins.

The blossom mutation results in an aspartic acid (D) to valine (V) substitution at position 233 (D223V) in the KLHL6 protein; amino acid 223 is within the BACK domain.

Please see the record cerulean for more information about Klhl6.

Putative Mechanism

KLHL6 has putative functions in B cell receptor-associated signal transduction and germinal center responses (2). Loss of KLHL6 expression results in impaired transitional B cell survival and differentiation (3). Klhl6-deficient (Klhl6^-/-) mice exhibited impaired B cell development past the immature stage, reduced numbers of B220+ and CD3- B cells in the spllen and peripheral blood, reduced numbers of follicular B cells in the lymph nodes, reduced spleen weight, spleen hypoplasia, impaired germinal center formation, and impaired memory IgG responses (2). Mice homozygous for an ENU-induced Klhl6 mutation (W267*) exhibited increased numbers of immature B cells, but reduced numbers of mature B cells (MGI).

Putative functions for KLHL6 in T cells has not been described. The role of KLHL6 in B cells is unknown, but it may contribute to cell proliferation and cell survival through interactions with proteins (e.g., HBXIP and Cul3) (3).

Primers

PCR Primer
blossom_pcr_F: TGGTACATCCTGGCTTCCTG
blossom_pcr_R: CTCACAGAAGCATTGAACCCGG

Sequencing Primer
blossom_seq_F: GACTTCAGGGCACTGTCTAATAAG
blossom_seq_R: GCATTGAACCCGGAAAATTGTATTGG

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 401 nucleotides is amplified (chromosome 16, - strand):

1   ctcacagaag cattgaaccc ggaaaattgt attggaatac tgaggctggc agacacacat
61  tctttggaca gtttaaagat gcaagtccaa agttacatca ttcaaaactt tgttcagatt
121 ctgaattccg aggagttcct tgaacttcct gtggacactt tgtaccacat cttgaggagt
181 gacgacctgt gtgtgactga agaggctcag gtgtttgaga ctgtgatgag ctgggtccgg
241 cacaaacaat cagaacgact ctgcctgctc ccttgtgtcc tcgagaatgt gcgtttacca
301 cttctggatc cgtggtactt cgtggagatg gttgaagcag accctcttat tagacagtgc
361 cctgaagtct tcccgctgct tcaggaagcc aggatgtacc a

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

References

  1. Gupta-Rossi, N., Storck, S., Griebel, P. J., Reynaud, C. A., Weill, J. C., and Dahan, A. (2003) Specific Over-Expression of Deltex and a New Kelch-Like Protein in Human Germinal Center B Cells. Mol Immunol. 39, 791-799.

  2. Kroll, J., Shi, X., Caprioli, A., Liu, H. H., Waskow, C., Lin, K. M., Miyazaki, T., Rodewald, H. R., and Sato, T. N. (2005) The BTB-Kelch Protein KLHL6 is Involved in B-Lymphocyte Antigen Receptor Signaling and Germinal Center Formation. Mol Cell Biol. 25, 8531-8540.

  3. Bertocci, B., Lecoeuche, D., Sterlin, D., Kuhn, J., Gaillard, B., De Smet, A., Lembo, F., Bole-Feysot, C., Cagnard, N., Fadeev, T., Dahan, A., Weill, J. C., and Reynaud, C. A. (2017) Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation. J Immunol. 199, 2408-2420.