Phenotypic Mutation 'tangyuan' (pdf version)
Alleletangyuan
Mutation Type critical splice donor site (1 bp from exon)
Chromosome9
Coordinate96,150,797 bp (GRCm38)
Base Change G ⇒ A (forward strand)
Gene Gk5
Gene Name glycerol kinase 5 (putative)
Synonym(s) C330018K18Rik, G630067D24Rik
Chromosomal Location 96,119,362-96,184,608 bp (+)
MGI Phenotype PHENOTYPE: Homozygous knockout does not result in an obvious skin phenotype and does not lead to alopecia. [provided by MGI curators]
Accession Number

NCBI Refseq: NM_177352.4; MGI: 2443336

Mapped Yes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000082313 ] [ENSMUSP00000112717 ] [ENSMUSP00000123594 ]   † probably from a misspliced transcript
SMART Domains Protein: ENSMUSP00000082313
Gene: ENSMUSG00000041440

DomainStartEndE-ValueType
low complexity region 4 20 N/A INTRINSIC
Pfam:FGGY_N 25 287 9e-50 PFAM
Pfam:FGGY_C 296 485 7.7e-35 PFAM
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000112717
Gene: ENSMUSG00000041440

DomainStartEndE-ValueType
low complexity region 4 20 N/A INTRINSIC
Pfam:FGGY_N 25 287 1.9e-49 PFAM
Pfam:FGGY_C 296 485 1.8e-35 PFAM
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000123594
Gene: ENSMUSG00000041440

DomainStartEndE-ValueType
low complexity region 4 20 N/A INTRINSIC
SCOP:d1bu6o1 24 56 1e-5 SMART
Predicted Effect probably benign
Meta Mutation Damage Score 0.702 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category
Phenotypequestion? Literature verified References
FACS CD4:CD8 - decreased
FACS CD44+ CD4 MFI - increased
FACS CD44+ CD4 T cells - increased
FACS CD44+ CD8 MFI - increased
FACS CD44+ CD8 T cells - increased
FACS CD44+ T cells - increased
FACS CD44+ T MFI - increased
FACS central memory CD8 T cells in CD8 T cells - increased
FACS naive CD8 T cells in CD8 T cells - decreased
skin/coat/nails 28607088
Candidate Explorer Status CE: excellent candidate; human score: 2; ML prob: 0.8264
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(20) : Chemically induced (other)(1) Gene trapped(17) Targeted(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01359:Gk5 APN 9 96137789 missense probably damaging 0.98
IGL01387:Gk5 APN 9 96177554 critical splice donor site probably null
IGL01771:Gk5 APN 9 96177435 missense probably damaging 0.97
IGL02253:Gk5 APN 9 96137771 missense probably damaging 1.00
IGL02380:Gk5 APN 9 96150480 missense possibly damaging 0.92
IGL02566:Gk5 APN 9 96129046 missense possibly damaging 0.56
IGL03137:Gk5 APN 9 96176292 splice site probably benign
IGL03256:Gk5 APN 9 96129053 missense probably damaging 1.00
IGL03326:Gk5 APN 9 96137839 critical splice donor site probably null
barrener UTSW 9 96129096 critical splice donor site probably null
glimpse UTSW 9 96181770 critical splice acceptor site probably null
homer UTSW 9 96140656 nonsense probably null
sean UTSW 9 96176237 nonsense probably null
stripped UTSW 9 96129053 missense probably damaging 1.00
toku UTSW 9 96140629 frame shift probably null
I1329:Gk5 UTSW 9 96140629 frame shift probably null
R0279:Gk5 UTSW 9 96174804 splice site probably benign
R0284:Gk5 UTSW 9 96181770 critical splice acceptor site probably null
R1134:Gk5 UTSW 9 96133407 missense probably benign 0.00
R1184:Gk5 UTSW 9 96150420 splice site probably benign
R1772:Gk5 UTSW 9 96150797 critical splice donor site probably null
R1781:Gk5 UTSW 9 96133455 missense possibly damaging 0.79
R3691:Gk5 UTSW 9 96129096 critical splice donor site probably null
R4213:Gk5 UTSW 9 96129053 missense probably damaging 1.00
R5015:Gk5 UTSW 9 96177417 critical splice acceptor site probably null
R5166:Gk5 UTSW 9 96174768 missense probably damaging 0.99
R5643:Gk5 UTSW 9 96140656 nonsense probably null
R5857:Gk5 UTSW 9 96119455 nonsense probably null
R5924:Gk5 UTSW 9 96150510 critical splice donor site probably null
R6109:Gk5 UTSW 9 96140610 missense probably benign 0.00
R6138:Gk5 UTSW 9 96176237 nonsense probably null
R6725:Gk5 UTSW 9 96155470 missense probably benign 0.01
R6812:Gk5 UTSW 9 96150749 missense probably damaging 0.99
R7065:Gk5 UTSW 9 96179056 missense probably damaging 1.00
R7182:Gk5 UTSW 9 96119526 missense possibly damaging 0.89
R7213:Gk5 UTSW 9 96145712 missense probably damaging 1.00
R7260:Gk5 UTSW 9 96119610 missense probably benign 0.10
U15987:Gk5 UTSW 9 96176237 nonsense probably null
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:46 PM by Anne Murray
Record Created 2015-03-11 12:15 PM by Zhao Zhang
Record Posted 2017-05-25
Phenotypic Description
Figure 1. Tangyuan mice exhibit hair loss.

Figure 2. Tangyuan mice exhibit increased frequencies of peripheral CD44+ 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 3. Tangyuan mice exhibit increased frequencies of peripheral central memory CD8 T cells in 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. Tangyuan mice exhibit increased expression of CD44 on peripheral blood CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 tangyuan phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1772, some of which showed hair loss (Figure 1). Some mice also showed increased frequencies of CD44+ CD8 T cells (Figure 2) and central memory CD8 T cells in CD8 T cells (Figure 3) in the peripheral blood. The expression of CD44 was increased on peripheral blood CD8 T cells (Figure 4).

Nature of Mutation

Figure 5. Linkage mapping of increased CD44+ CD8 T cell frequency using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 107 mutations (X-axis) identified in the G1 male of pedigree R1772. 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 114 mutations. Among these, only one affected a gene with known effects on hair loss, Gk5. The mutation in Gk5 was presumed to be causative because the tangyuan hair loss phenotype mimics other known alleles of Gk5 (see the entries for toku and barrener). The Gk5 mutation is a G to A transition at base pair 96,150,797 (v38) on chromosome 9, or base pair 31,454 in the GenBank genomic region NC_000075 in the donor splice site of intron 9. The strongest association was found with a recessive model of inheritance to the normalized frequency of CD44+ CD8 T cells, wherein two variant homozygotes departed phenotypically from three homozygous reference mice and 13 heterozygous mice with a P value of 1.953 x 10-5 (Figure 5).

 

The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in skipping of the 61-nucleotide exon 9 (out of 16 total exons), resulting in a frame shifted protein product beginning after amino acid 256 of the protein and terminating after the inclusion of 19 aberrant amino acids.

 

          <--exon 8      <--exon 9 intron 9-->               <--exon 10-->

31155 ……AAGGATACCAG ……GTTGTTGCCTTG gtaagtagaaggcatt…… GTTGGTGACCAGC……CGGGAGATGTGA

254   ……-K--D--T--S ……-V--V--A--L-                    --L--V--T--S-……-R--E--M--*-
         correct       deleted                               aberrant

 

Genomic numbering corresponds to NC_000075. The donor splice site of intron 9, which is destroyed by the tangyuan mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Protein Prediction

Figure 6. Domain organization of GK5. The location of the tangyuan mutation is indicated. Domain information is from SMART and UniProt. Additional mutations in GK5 are noted. Click on each mutation to view more information.

The glycerol kinase 5 (putative) (Gk5) gene encodes the 534 amino acid (aa) GK5 protein. The protein domains and function of GK5 have not been documented. SMART predicts that this uncharacterized protein contains two domains that are found in the FGGY family of carbohydrate kinases. The FGGY kinases contain conserved motifs at both the N- and C-termini (Figure 6; aa 25-287 and aa 396-416 in GK5, respectively; SMART). The FGGY_N and FGGY_C termini are structurally similar and adopt a ribonuclease H-like fold (1;2). Between the FGGY_N and FGGY_C domains is a catalytic cleft where the sugar substrate and ATP bind (3).  The tangyuan mutation is predicted to result in skipping of exon 9, resulting in a frame shifted protein product beginning after amino acid 256 of the protein and terminating after the inclusion of 19 aberrant amino acids.

 

For more information about Gk5, please see the entry for toku.

Putative Mechanism

The over 4,000 members of the FGGY family phosphorylate sugar substrates in an ATP-dependent manner (3). Similar to glycerol kinase, GK5 is proposed to be involved in ATP binding, phosphotransferase activity, and glycerol kinase activity. GK5 is necessary for hair growth, and functions in the regulation of SREBP-1/-2-mediated cholesterol production in the skin (4). The buildup of sterol precursors in the sebocytes results in defects in hair follicle development and homeostasis as observed in the tangyuan mice (4).

Primers PCR Primer
tangyuan_pcr_F: ACTTTATGGGCTACGCGAAAAGCAG
tangyuan_pcr_R: TGAAATATAAGCAGCAGTGACCCCTTG

Sequencing Primer
tangyuan_seq_F: TACGCGAAAAGCAGTTTGCC
tangyuan_seq_R: ggcaaatgctttaacactaacac
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 649 nucleotides is amplified (chromosome 9, + strand):


1   actttatggg ctacgcgaaa agcagtttgc cggattttag ggtgagtgcg tccagagcta
61  caactaactc tttcatcgat ctcatttgaa gctataattt tggatcagtg gatgagaaga
121 tatttggcgt gcctatacca gttgttgcct tggtaagtag aaggcattgg ggtgggggtg
181 taagagctaa ataattggat agttggggcg agctttcaga gaaacagaag gtctttagta
241 gaataatgcc tgagccaggg gtggtggctc ccagctttac tcccagtagt cactcaaggg
301 cacaggcagg caggttttgt gagtttgagg ccagcctgat ctacacagca agttccaagg
361 ctgccagagc cacacaggta ggccctactg ttcttagtgt tagtgttaaa gcatttgcca
421 ccaggtacaa ggccctgggc tcaatatcag cactaaagaa gaaacacaca ttctgcctgg
481 tatctctttt actttctttt aagttaaaag ggtcgtgtat ataatgagaa gcatttccta
541 tataaggaaa ctgtggcatt ttctaaagat gctgtgttct gagtaaaaga acacatctgt
601 agatgcttga tttgaaaaac agcaaggggt cactgctgct tatatttca


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

References
Science Writers Anne Murray
Illustrators Katherine Timer
AuthorsZhao Zhang, Doan Dao, Hexin Shi, Lei Sun, Jianhui Wang, Ying Wang, Bruce Beutler