Phenotypic Mutation 'Parula' (pdf version)
AlleleParula
Mutation Type missense
Chromosome16
Coordinate19,775,793 bp (GRCm39)
Base Change A ⇒ T (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]
Accession Number

NCBI RefSeq: NM_183390; MGI:2686922

MappedYes 
Amino Acid Change Valine changed to Aspartic acid
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000053023] [ENSMUSP00000130755]
AlphaFold Q6V595
SMART Domains Protein: ENSMUSP00000053023
Gene: ENSMUSG00000043008
AA Change: V255D

DomainStartEndE-ValueType
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 possibly damaging

PolyPhen 2 Score 0.555 (Sensitivity: 0.88; Specificity: 0.91)
(Using ENSMUST00000058839)
SMART Domains Protein: ENSMUSP00000130755
Gene: ENSMUSG00000043008

DomainStartEndE-ValueType
Pfam:BTB 60 98 1.2e-9 PFAM
Predicted Effect noncoding transcript
Meta Mutation Damage Score 0.4807 question?
Is this an essential gene? Probably nonessential (E-score: 0.072) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(6) : Chemically induced (ENU)(1) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00788:Klhl6 APN 16 19775812 missense probably benign 0.00
IGL01465:Klhl6 APN 16 19801572 missense probably damaging 0.98
IGL01831:Klhl6 APN 16 19772235 missense probably damaging 1.00
IGL01971:Klhl6 APN 16 19768276 missense probably damaging 0.99
IGL02532:Klhl6 APN 16 19775832 missense possibly damaging 0.84
IGL03113:Klhl6 APN 16 19776001 missense possibly damaging 0.68
IGL03290:Klhl6 APN 16 19765887 missense probably benign 0.44
Ascension UTSW 16 19765848 missense probably damaging 1.00
besmirched UTSW 16 19768197 splice site probably null
blau UTSW 16 19775755 missense probably damaging 1.00
blossom UTSW 16 19775889 missense probably damaging 1.00
Breech UTSW 16 19766984 missense probably benign 0.43
cerulean UTSW 16 19775968 nonsense probably null
cobalt UTSW 16 19775772 missense probably damaging 1.00
grossbeak UTSW 16 19768201 missense probably null 1.00
heights UTSW 16 19775778 missense probably damaging 0.98
Lazuli UTSW 16 19775716 frame shift probably null
sideways UTSW 16 19776018 missense probably damaging 0.99
torres_del_paine UTSW 16 19766877 missense probably damaging 1.00
turquoise UTSW 16 19801546 missense probably damaging 1.00
IGL03046:Klhl6 UTSW 16 19801639 missense probably benign
R0265:Klhl6 UTSW 16 19766984 missense probably benign 0.43
R0496:Klhl6 UTSW 16 19775716 frame shift probably null
R0497:Klhl6 UTSW 16 19775716 frame shift probably null
R0540:Klhl6 UTSW 16 19775764 missense possibly damaging 0.95
R0541:Klhl6 UTSW 16 19768197 splice site probably null
R0554:Klhl6 UTSW 16 19772343 missense probably damaging 0.96
R0607:Klhl6 UTSW 16 19775764 missense possibly damaging 0.95
R0636:Klhl6 UTSW 16 19766823 splice site probably benign
R0670:Klhl6 UTSW 16 19768309 missense possibly damaging 0.92
R1477:Klhl6 UTSW 16 19784727 missense probably benign 0.00
R1510:Klhl6 UTSW 16 19765848 missense probably damaging 1.00
R1547:Klhl6 UTSW 16 19784832 missense probably benign
R1747:Klhl6 UTSW 16 19765778 missense probably benign 0.40
R1871:Klhl6 UTSW 16 19775793 missense possibly damaging 0.56
R1966:Klhl6 UTSW 16 19801572 missense probably damaging 0.98
R2058:Klhl6 UTSW 16 19801681 missense probably benign
R4466:Klhl6 UTSW 16 19776018 missense probably damaging 0.99
R4645:Klhl6 UTSW 16 19765897 missense probably damaging 1.00
R4690:Klhl6 UTSW 16 19776034 missense probably benign 0.44
R4824:Klhl6 UTSW 16 19775778 missense probably damaging 0.98
R4833:Klhl6 UTSW 16 19775889 missense probably damaging 1.00
R4835:Klhl6 UTSW 16 19775783 missense probably benign 0.07
R5001:Klhl6 UTSW 16 19765741 makesense probably null
R5475:Klhl6 UTSW 16 19766877 missense probably damaging 1.00
R5700:Klhl6 UTSW 16 19775968 nonsense probably null
R5867:Klhl6 UTSW 16 19801570 missense probably benign 0.37
R5910:Klhl6 UTSW 16 19775844 missense probably benign 0.04
R6992:Klhl6 UTSW 16 19772337 missense probably damaging 1.00
R7082:Klhl6 UTSW 16 19801633 missense probably benign 0.00
R7262:Klhl6 UTSW 16 19801546 missense probably damaging 1.00
R7314:Klhl6 UTSW 16 19775755 missense probably damaging 1.00
R7464:Klhl6 UTSW 16 19775863 missense possibly damaging 0.58
R7688:Klhl6 UTSW 16 19765881 missense probably damaging 1.00
R7957:Klhl6 UTSW 16 19768201 missense probably null 1.00
R8319:Klhl6 UTSW 16 19775940 missense possibly damaging 0.74
R8460:Klhl6 UTSW 16 19775781 missense probably damaging 1.00
R8853:Klhl6 UTSW 16 19765979 missense possibly damaging 0.52
R9046:Klhl6 UTSW 16 19765803 missense probably damaging 1.00
R9160:Klhl6 UTSW 16 19775772 missense probably damaging 1.00
Z1176:Klhl6 UTSW 16 19772424 missense probably damaging 1.00
Z1177:Klhl6 UTSW 16 19801711 nonsense probably null
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:33 PM by Diantha La Vine
Record Created 2018-12-09 12:50 PM by Bruce Beutler
Record Posted 2018-12-18
Phenotypic Description

Figure 1. Parula 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 2. Parula 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 3. Parula 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.
Figure 4. Parula mice exhibit increased frequencies of peripheral B1a cells. Flow cytometric analysis of peripheral blood was utilized to determine B1a 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 Parula phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1871, some of which showed increased frequencies of T cells (Figure 1), CD4+ T cells (Figure 2), CD8+ T cells (Figure 3), and B1a cells (Figure 4) in the peripheral blood.

Nature of Mutation

Figure 5. Linkage mapping of the increased T cell frequency using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 81 mutations (X-axis) identified in the G1 male of pedigree R1871. 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 81 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Klhl6:  a T to A transversion at base pair 19,957,043 (v38) on chromosome 16, or base pair 26,007 in the GenBank genomic region NC_000082 encoding Klhl6. The strongest association was found with an additive model of inheritance to the normalized frequency of T cells, wherein four variant homozygotes and seven heterozygous mice departed phenotypically from nine homozygous reference mice with a P value of 7.53 x 10-7 (Figure 5).  

The mutation corresponds to residue 810 in the mRNA sequence NM_183390 within exon 3 of 7 total exons.


 

794 TGCCTGCTCCCTTGTGTCCTCGAGAATGTGCGT

250 -C--L--L--P--C--V--L--E--N--V--R-

The mutated nucleotide is indicated in red. The mutation results in a valine to aspartic acid substitution at position 255 (V255D) in the KLHL6 protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.555).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 6. Domain structure of the KLHL6 protein. KLHL6 is a member of the Kelch-like family. The Parula mutation (V255D) 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) (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 Parula mutation results in a valine to aspartic acid substitution at position 255 (V255D); amino acid 255 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 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) .

Primers PCR Primer
Parula_pcr_F: AGAGTCCCTGGATAATGCCTTAC
Parula_pcr_R: GGACAGTTTAAAGATGCAAGTCC

Sequencing Primer
Parula_seq_F: TGGATAATGCCTTACATTCCCAAC
Parula_seq_R: AGATGCAAGTCCAAAGTTACATC
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 414 nucleotides is amplified (chromosome 16, - strand):


1   ggacagttta aagatgcaag tccaaagtta catcattcaa aactttgttc agattctgaa
61  ttccgaggag ttccttgaac ttcctgtgga cactttgtac cacatcttga ggagtgacga
121 cctgtgtgtg actgaagagg ctcaggtgtt tgagactgtg atgagctggg tccggcacaa
181 acaatcagaa cgactctgcc tgctcccttg tgtcctcgag aatgtgcgtt taccacttct
241 ggatccgtgg tacttcgtgg agatggttga agcagaccct cttattagac agtgccctga
301 agtcttcccg ctgcttcagg aagccaggat gtaccacctt tctggcaatg aggtgagttg
361 gacttgagag gtatttcatt ttgttgggaa tgtaaggcat tatccaggga ctct


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