Phenotypic Mutation 'Tootsie' (pdf version)
AlleleTootsie
Mutation Type missense
Chromosome11
Coordinate5,859,150 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Gck
Gene Name glucokinase
Synonym(s) Gls006, hexokinase 4, HK4, MODY2, Hlb62
Chromosomal Location 5,850,820-5,900,081 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Hexokinases phosphorylate glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways. Alternative splicing of this gene results in three tissue-specific forms of glucokinase, one found in pancreatic islet beta cells and two found in liver. The protein localizes to the outer membrane of mitochondria. In contrast to other forms of hexokinase, this enzyme is not inhibited by its product glucose-6-phosphate but remains active while glucose is abundant. Mutations in this gene have been associated with non-insulin dependent diabetes mellitus (NIDDM), maturity onset diabetes of the young, type 2 (MODY2) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). [provided by RefSeq, Apr 2009]
PHENOTYPE: Targeted disruption of this gene causes mild hyperglycemia in heterozygous mice and extreme hyperglycemia and embryonic to postnatal lethality in homozygous mice. Hyperglycemic knock-out or ENU-induced mutants may show reduced body weight and liver glycogen level, hepatic steatosis, and glucosuria. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_010292 (variant 1), NM_001287386 (variant 2); MGI:1270854

MappedYes 
Amino Acid Change Methionine changed to Lysine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000099984] [ENSMUSP00000105447] [ENSMUSP00000105448]
AlphaFold P52792
SMART Domains Protein: ENSMUSP00000099984
Gene: ENSMUSG00000041798
AA Change: M139K

DomainStartEndE-ValueType
Pfam:Hexokinase_1 10 217 4.3e-80 PFAM
Pfam:Hexokinase_2 219 458 1.3e-100 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.633 (Sensitivity: 0.87; Specificity: 0.91)
(Using ENSMUST00000102920)
SMART Domains Protein: ENSMUSP00000105447
Gene: ENSMUSG00000041798
AA Change: M139K

DomainStartEndE-ValueType
Pfam:Hexokinase_1 10 217 1e-79 PFAM
Pfam:Hexokinase_2 219 458 7.8e-101 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.633 (Sensitivity: 0.87; Specificity: 0.91)
(Using ENSMUST00000109822)
SMART Domains Protein: ENSMUSP00000105448
Gene: ENSMUSG00000041798
AA Change: M139K

DomainStartEndE-ValueType
Pfam:Hexokinase_1 15 216 1.9e-74 PFAM
Pfam:Hexokinase_2 221 455 2.2e-79 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.633 (Sensitivity: 0.87; Specificity: 0.91)
(Using ENSMUST00000109823)
Meta Mutation Damage Score 0.8231 question?
Is this an essential gene? Essential (E-score: 1.000) question?
Phenotypic Category Autosomal Dominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(35) : Chemically induced (ENU)(19) Gene trapped(5) Targeted(11)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01624:Gck APN 11 5853106 missense possibly damaging 0.67
IGL01647:Gck APN 11 5854472 missense probably damaging 0.97
IGL03145:Gck APN 11 5859093 missense probably damaging 0.99
Grahamcracker UTSW 11 5852165 missense probably damaging 1.00
R0139:Gck UTSW 11 5860370 missense probably damaging 1.00
R0139:Gck UTSW 11 5859139 nonsense probably null
R0691:Gck UTSW 11 5856691 missense probably damaging 1.00
R1829:Gck UTSW 11 5860984 missense probably damaging 0.97
R1866:Gck UTSW 11 5853253 missense probably benign 0.02
R1868:Gck UTSW 11 5852165 missense probably damaging 1.00
R1992:Gck UTSW 11 5856515 missense probably damaging 1.00
R3885:Gck UTSW 11 5860318 missense probably damaging 1.00
R4179:Gck UTSW 11 5860295 missense probably benign 0.43
R4888:Gck UTSW 11 5859150 missense possibly damaging 0.63
R7034:Gck UTSW 11 5851747 missense probably damaging 1.00
R7155:Gck UTSW 11 5899705 start gained probably benign
R7548:Gck UTSW 11 5852040 missense
R8039:Gck UTSW 11 5860301 missense probably benign 0.12
R8891:Gck UTSW 11 5851733 missense probably damaging 1.00
R9100:Gck UTSW 11 5856516 missense probably damaging 1.00
R9101:Gck UTSW 11 5856516 missense probably damaging 1.00
R9102:Gck UTSW 11 5856516 missense probably damaging 1.00
R9116:Gck UTSW 11 5854377 missense possibly damaging 0.71
R9370:Gck UTSW 11 5852244 missense possibly damaging 0.78
R9420:Gck UTSW 11 5899553 critical splice donor site probably null
R9536:Gck UTSW 11 5852307 missense possibly damaging 0.92
Z1176:Gck UTSW 11 5856526 missense probably damaging 1.00
Z1177:Gck UTSW 11 5860958 missense possibly damaging 0.81
Mode of Inheritance Autosomal Dominant
Local Stock
Repository
Last Updated 2019-09-04 9:41 PM by Anne Murray
Record Created 2016-12-16 8:19 PM
Record Posted 2018-09-13
Phenotypic Description

Figure 1. Tootsie mice exhibited high blood glucose levels 30 minutes after glucose challenge. 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 Tootsie phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4888, some of which showed high blood glucose levels 30 minutes after glucose challenge (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the high blood glucose levels 30 minutes after glucose using a dominant model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 154 mutations (X-axis) identified in the G1 male of pedigree R4888. 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 154 mutations. The hyperglycemia phenotype was linked by continuous variable mapping to a mutation in Gck: a T to A transversion at base pair 5,909,150 (v38) on chromosome 11, or base pair 40,932 in the GenBank genomic region NC_000077 encoding Gck. Linkage was found with an additive/dominant model of inheritance, wherein 10 heterozygous mice departed phenotypically from eight homozygous reference mice with a P value of 3.116 x 10-5 (Figure 2); no homozygous variant mice were observed in pedigree R4888.   

The mutation corresponds to residue 898 in the mRNA sequence NM_010292 within exon 4 of 10 total exons.

882 CTGGACAAGCATCAGATGAAACACAAGAAACTA

134 -L--D--K--H--Q--M--K--H--K--K--L-

The mutated nucleotide is indicated in red. The mutation results in a methionine to lysine substitution at amino acid 139 (M139K) in the GCK protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.633).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Domain organization of GCK. The location of the Tootsie mutation is indicated. The residues involved in ATP and glucose binding are indicated. The small and large domains refer to the corresponding domains in the crystal structures shown on the Grahamcracker entry. Domain information is from SMART and UniProt. This image is interactive; click to view additional mutations in Gck.

Glucokinase [GCK; alternatively, hexokinase 4 (HK4)] is a member of the hexokinase family (Figure 3). Alternative splicing of Gck generates three tissue-specific 465-amino acid GCK isoforms: GCK-B1, GCK-B2, and GCK-L1 (1-4). GCK-B1 and GCK-B2 are specific to pancreatic β-cells, while GCK-L1 is specific to the liver. At present, it is unknown whether it is the B1 or B2 isoform (or both) that generate the pancreatic GCK enzymatic activity. The GCK-L1 isoform differs from the GCK-B isoforms within the 16 N-terminal amino acids; the functional significance of the N-terminus of all of the isoforms is unknown. The crystal of GK(Δ1-11) in complex with glucose and compound A, an activator, folded into a large and small subdomain corresponding to amino acids 67-203 and 204-443 in mouse GCK, respectively (5). The large and small subdomains are separated by a deep cleft which forms the active site (5).

The Tootsie mutation results in a methionine to lysine substitution at amino acid 139 (M139K) in the GCK protein. M139 is within the GK small subdomain.

Please see the record Grahamcracker for more information about Gck.

Putative Mechanism

GCK is one of four members of the hexokinase family. In the gut, GCK may promote the secretion of enteroincretins, which are hormones that stimulate a decrease in blood glucose levels (e.g., GLP-1) (6). In the brain, GCK may assist in the regulation of feeding behavior and counter regulatory responses by functioning as a glucose sensor (6;7). In the liver and pancreas, GCK is a component of the ‘glucose sensor’ that regulates plasma glucose levels.

Mutations in GCK are linked to gestational diabetes mellitus [OMIM: #125851; (8)], reduced birth rate (9), late onset noninsulin-dependent diabetes mellitus [OMIM: #125853; (10)], permanent neonatal diabetes mellitus [OMIM: #606176; (11-13)], familial hyperinsulinemic hypoglycemia 3 [OMIM: #602485; (14;15)], and type II maturity-onset diabetes of the young [MODY 2 (alternatively, GCK-MODY); OMIM: #125851; (12;16-18)]. All of the above-mentioned conditions are the result of variable degrees of glucose intolerance due to impaired glucose-responsive insulin secretion.

Beta cell-specific Gck heterozygous (β-Gck+/-) mice exhibit moderate hyperglycemia and defective insulin secretion in response to glucose (19;20). Pancreatic beta cells from the β-Gck+/- mice exhibited impaired glucose sensitivity that worsened with age (21). β-Gck+/- mice fed a high-fat diet exhibited reduced beta cell replication and beta cell hyperplasia compared to wild-type mice (22). In addition, islets from the β-Gck+/- mice had diminished expression of IRS-2 compared to that in wild-type mice (22). In the liver, GCK function mediates hepatic glucose uptake, the synthesis and subsequent storage of glycogen in the liver, and the regulation of glucose-responsive genes (19;23). Hepatocyte-specific conditional Gck knockout (Liver-Gck-/- ) mice were mildly hyperglycemic, but exhibited defects in glycogen synthesis, glucose turnover rates during hyperglycemic clamp, and impaired insulin secretion in response to glucose (19;24;25). Hepatocyte-specific Gck knockdown in obesity-prone mice attenuated weight gain with a concomitant increase in adaptive thermogenesis (26). Gck knockout (Gck-/-) mice are not viable and die from approximately embryonic day (E) 9 until shortly before birth from severe hyperglycemia (23;27;28). Gck heterozygous (Gck+/-) mice survive, but exhibit reduced islet GCK activity and subsequent elevation in fasting blood glucose levels (27-29). After hyperglycemic clamp, the Gck+/- mice exhibited reduced glucose tolerance and defective liver glucose metabolism (27;29). In addition, Gck+/- mice exhibited reduced fertility, increased levels of plasma corticosterone, increased food intake, and hypothalamic gene expression (e.g., increased hypothalamic neuropeptide Y mRNA and reduced hypothalamic proopiomelanocortin mRNA) (23). ENU-induced mutations in Gck resulted in variable reduced viability, reduced GCK activity, and subsequent impaired glucose-responsive insulin secretion (30-33). Several homozygote ENU-induced models (GckGENA348/GENA348 and GckD217VD217V/D217V) exhibited increased viability (e.g., 5-12 weeks of age) compared to the Gck-/- mice (31;34). However, the ENU-induced mutant Munich GckM210R/M210R mice exhibited growth retardation and perinatal lethality (33). All heterozygote ENU-induced models exhibited elevated plasma glucose levels, impaired glucose tolerance, and reduced glucose-induced insulin secretion compared to wild-type mice (31;33;34).

Similar to other ENU-induced mutants, the Tootsie mice exhibit impaired glucose tolerance indicating that the mutation may result in almost complete loss of GCKTootsie function.

Primers PCR Primer
Tootsie_pcr_F: ACAGTTGCAATCCCCACTCG
Tootsie_pcr_R: GTCCCTAAGAGTGTGGATGAG

Sequencing Primer
Tootsie_seq_F: AATCCCCACTCGGCATGG
Tootsie_seq_R: AAGATGCTCTAAGTGGGAATTTGG
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 417 nucleotides is amplified (chromosome 11, - strand):


1   gtccctaaga gtgtggatga ggggagaaga tgctctaagt gggaatttgg tgaggcagaa
61  ctggagctgc ctcaggggtc aaatgtctta acatctccaa gagacacttt ggtacagttc
121 ctggggagag agaactgcta ctgtcccagc cctgacctaa tgccacactg gtgcaactcc
181 tagctctttg actacatctc tgagtgcatc tctgacttcc tggacaagca tcagatgaaa
241 cacaagaaac tacccctggg cttcaccttc tccttccctg taaggcacga agacatagac
301 aaggtgagca ggtggaggag agaggagatg aatgggtgga aacacttggg aagaactctg
361 ccgagactgc cttcagttgc agctccccaa cccatgccga gtggggattg caactgt


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
AuthorsEmre Turer and Bruce Beutler