The Besmirched phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R0541, some of which showed reduced B220 expression on peripheral blood B cells (Figure 1).

Whole exome HiSeq sequencing of the G1 grandsire identified 68 mutations. The B220 phenotype was linked by continuous variable mapping to mutations in two genes on chromosome 16: Spidr and Klhl6. The mutation in Klhl6 was presumed causative as the immune phenotype observed in the Besmirched mice mimics that of other Klhl6 mutant alleles. The Klhl6 mutation is an A to T transversion at base pair 19,949,447 (v38) on chromosome 16, or base pair 33,603 in the GenBank genomic region NC_000082 within the splice donor site of intron 5 (3-base pairs from exon 5). The strongest association was found with a recessive model of inheritance to the CD4+ T cell phenotype, wherein two variant homozygotes mice departed phenotypically from six homozygous reference mice and 10 heterozygous with a P value of 6.508 x 10^-5 (Figure 3).  

The effect of the mutation at the cDNA and protein levels has not been examined, but the mutation is predicted to result in the use of a cryptic site in intron 5. The mutation would result in an 85-base pair insertion of intron 5, causing a frame-shifted protein product beginning after amino acid 381 of the protein (which is normally 619 amino acids in length), and termination after the inclusion of six aberrant amino acids.

           <--exon 4      <--exon 5 intron 5-->                     exon 6-->

1175 ……GTCTACATATCAG ……TGCTGGTCAGAG gtatgggagatggtt……ctctgcctccacag GCTGCACCCCTCC……

377  ……-V--Y--I--S-- ……-C--W--S--E-                                 -A--A--P--L--……

The donor splice site of intron 5, which is destroyed by the Besmirched mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

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 4) (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 Besmirched mutation within the donor splice site of intron 5 is predicted to result in a frame-shifted protein product beginning after amino acid 381 of the protein.

Please see the record cerulean for more information about Klhl6.

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 spleen 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). The phenotype observed in the Besmirched mice indicate loss of KLHL6-associated function.

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

1   cttgtaggtg gcaaagaaac acagcatgat gtttggaagt acaattcctc catcaacaaa
61  tggattcaga tcgaatattt aaacataggt cgctggaggc ataagatggt cgtattgggt
121 ggcaaagtct atgtacttgg gggttttgat ggcttacaaa gaatcaacaa tgtggagaca
181 tatgacccct tccacaactg ctggtcagag gtatgggaga tggtttctat aagtaatttc
241 aacaaaagta ctgcatgtgg tggtggcagg ctttttgcct actttgttcc ttcaggtgct
301 ttgaccttct ctcttcctag tagccatggc atcaacgtta ctaatctaga tccagtaata
361 acacccgtct agatgacagg gctctgtata ggcattgttc tgtgctagcc agagtctcag
421 ctatagaggg aaggctgagc ctatccctaa gatggtttag ctctgttggt ggcatatcct
481 ggctgagtct gtctcctcag atgct

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