1. T2D- A complex trait Identification, mapping and cloning of new type 2 diabetes models from ENU mutagenesis Medical Research Council, Mammalian Genetics Unit, UK

2. Aim
Resolving complex genetic determinants of common disease phenotypes
ENU mutagenesis to create monogenic diabetic traits
Creation of dominant alleles of any gene that can be mutated to yield a diabetic or sub-diabetic phenotype
ENU mutagenesis to modify insulin resistance and create diabetic traits (sensitised/modifier screen)
Analysis of genes and the pathways in which they lie

3. Dominant monogenic ENU models

6. Cloning: Gena 348 - Glucokinase Exon 9 A-T transversion
ccaggaccctcagtgacttccctccctaataccgcccgcagCGACTCTGGGGACCGAAGGCAGATCCTTAACATCCTGAGCACTCTGGGCCTTCGACCCTCTGTCGCCGACTGCGACATTGTGCGCCGTGCCTGTGAAAGCGTGTCCACTCGCGCCGCCCACATGTGCTCAGCAGGACTAGCGGGGGTCATAAATCGCATGCGCGAAAGCCGCAGTGAGGACGTGATGCGCATCACGGTGGGCGTGGATGGCTCCGTGTACAAGCTGCACCCGAGgtcagcctccaccttcgctcgcgctccaacctcccgggctccaagggaggctccaagggcgcggctttcaactccaggctttt
Isoleucine ATT Phenylalanine TTT
CH2 CH
CH CH C
HC CH
CH3
non polar- hydrophobic

7. BC3 males 14wks, N=7 +/+, N=5 +/-P=

8. Male Female
Student T-Test (2-tailed) p value =

9. Homozygous viable
Very low GK activity G

10. Summary: GENA 348 GK, Isoleucine>Phenylalanine
GENA 389 Chr 11, GK splice mutation
GENA 396 Chr 11, GK splice mutation
GENA 387 Chr 11, GK splice mutation
GENA 263 Chr 6 (No GK poly)
GENA 394 Multiple linkages (No GK poly)
GENA 392 Chr 4- Dyslipidaemia mutant

11. Mutations can be mapped and cloned
ENU mutagenesis can be used to make new diabetic and sub-diabetic models
MODY models
Other genes
Mutations can be mapped and cloned
It should be possible to identify novel genes and/or provide new information on known diabetes genes and their pathways

12. Sensitised Screen
Michelle Goldsworthy

13. Sensitised/Modifier screens
Genetic backgrounds that predispose to a phenotype (T2D)
Insulin resistance but not overt diabetes
Screen for ENU mutations that result in a more severe IGT or full diabetic phenotype
Independent effect (as simple dominant screen)
Additivity of effect of the new ENU allele
Gene/ pathway interaction
Modify a phenotype
ENU mutations that suppress a phenotype
ENU mutations that exacerbate a phenotype

14. Dominant or Partially Sensitised
ENU mutagenised IR+/- or IRS-1+/- male mice
Backcrossed to C3H females
F1 mice assayed for impaired glucose tolerance (IPGTT)
Inheritance testing
Sensitised phenotype
Dominant or Partially Sensitised
Phenotype
IGT/10
IGT/3 IGT/4 IGT/6 ?
(new lines)

15. Sensitised screen 1292 F1 individuals generated
20 F1 individuals entered inheritance testing
14 IR +/-
6 IRS-1 +/-
Status:
4 lines segregation confirmed (IGT/3, 4, 6 & 10).
6 lines no robust phenotype inherited
10 lines still being tested

17. IGT/10 70 BC1 mice generated 43 males and 27 females: BC2 mice
Mixture of assays
Male segregating glycosuria
43 males and 27 females: BC2 mice
testing at 2 weekly intervals
Glycosuria segregates in males only
Peristant glycosuria present in IR +/- only

19. Genotyping
Minimal set of 32 BC1 and BC2 individuals selected for genotyping
Linkage obtained on Chromosome 14
- (LOD 4.3)
- Additional animals
genotyped (LOD 8.9)

21. IR +/+, Chr14 Het
IR +/-, Chr14 C3H
IR +/-, Chr14 Het
12, 14, 16, 18, 20 weeks of age

22. Future Work Fine Mapping (Further Backcrossing)
Candidate gene sequencing
Full pathology
9 mice (3 each group) now being analysed
Clamps
Model of Diabetic nephropathy?
Founder male shows an increase in mesangial matrix area (63.6% vs 52.7% P= )

23. P=1.7E-05
P=2.2E-05

25. P 3.15E-06
P 0.004

26. IGT/4 summary
Impaired glucose tolerance segregating in BC1 and BC2 populations
50% IR+/- exhibit IGT
25% IR+/+ exhibit IGT
Genotyping completed
Linkage chromosome 13 (BC1)
Additional fine mapping underway BC2 and BC3 individuals (E.H)
Candidate genes (E.H)

27. P= 1.8E-10
P= 1.4E-8

28. IGT/6 Summary Inherited impaired glucose tolerance
Significantly heavier than controls
Genotyping commenced BC1 individuals (Q.A)
Histological analysis
- Islet cell hyperplasia
- Nonalcoholic Steatohepatitis (N.A.S.H) (Q.A)

29. Future work Genotyping Fine mapping/candidate gene analysis
- IGT/3 (A.H)
- IGT/6 (Q.A)
Fine mapping/candidate gene analysis
- IGT/4 (E.H, M.G) IGT/10 (M.G)
Histology/microarray
- Islet hyperplasia IGT/3, IGT/6 (A.H, V.M)
- Kidney IGT/10 (C.P)
- NASH (Liver) IGT/6 (Q.A)
New Lines (J.Q, M.G)

30. Prospects- complementary approaches to complex traits
Dissect diseases with a complex genetic component into genes and pathways using mutagenesis approaches
Random mutagenesis may yield new genes and pathways
Sensitised screens may allow particular pathways or disease processes to be targetted

31. Further Prospects Generation of allelic series in genes of interest
Reconstitution of defined polygenic models
Combination of phenotypes i.e. metabolic syndrome models
Study of disease complications
Diabetic kidney
Diabetic heart

32. Acknowledgements Liz Bentley Toni Bell Catriona Paul
Deen Quwailid
Siobhan Mansell
Vesna Mijat
Alison Haynes
Charlotte East
Alison Hugill
Julie Quaterman
Ayo Toye
Roger Cox
Toni Bell
Jo Clay
Debbie Ritson
Jo Madge
Lee Moir
Quentin Anstee (IC London)
Emma Horner (Etiologics)
Medical Research Council