1. Diversity Outbred Mice
Daniel Gatti, Ph.D
May 2016
International Behavioural and Neural Genetics Society

2. Inbred Mice are useful models of disease
Homozygous across their entire genome.
All mice within a strain are genetically identical.
Reproducible models of disease.
A/J: high incidence of spontaneous lung adenomas
C3H/HeJ: high incidence of spontaneous hepatomas
C57BL/6J: a.k.a. “The Mouse”

3. Is an inbred mouse a good model for the genetically diverse human population?=
C57BL/6J =

4. Genetically diverse mouse models may improve translational relevance

5. Laboratory mice are derived from three major sub-species
Silver, Bonhomme

6. Classical inbred lines have limited genetic diversity
M.m.domesticus
M.m.musculus
Wild Caught
M.m.castaneus
Wild Derived Inbred
Classical Inbred
Yang et.al., Nat.Gen., 2011

7. Multiparent crosses can increase genetic diversity
Use 8 inbred founder lines.
Select founder lines to increase genetic diversity.
Morgan & Welsh, Mamm.Gen., 2015

8. Eight inbred founder strains of Collaborative Cross & Diversity Outbred
A/J
C57BL/6J
129S1/SvImJ
NOD/ShiLtJ
NZO/HlLtJ
CAST/EiJ
PWK/PhJ
WSB/EiJ A B C D E F G H
Classical Inbred
Wild Derived Inbred

9. Collaborative Cross (CC) mice are inbred strains derived from 8 inbred founders

10. Collaborative Cross mice
Available from Univ. of North Carolina. ~ 70 strains available. CC Genomes are a homozygous mosaic of the 8 founders. All mice within one strain are genetically identical. Useful for repeated measures of traits in the same genetic background.
CC Genome
Morgan & Welsh, Mamm.Gen.,2015

11. Diversity Outbred (DO) mice are a populations derived from the same 8 inbred founders

12. Diversity Outbred mice
Available from The Jackson Laboratory.
Contain 45 million SNPs.
Humans have ~15 mil. common SNPs.
Maintained by random mating.
Each mouse is genetically unique (like humans).
Useful for genome wide association mapping in mice.
Useful as a model of human genetic variation.

13. Diversity Outbred (DO) mice are an outbred population derived from 8 inbred founders
40.4 million SNPs
Phifer-Rixey & Nachman, Elife, Apr., 2015

14. 1000 Genomes Project
Phase III: 2,504 humans : 84.8 million SNPs

15. There are more SNPs in 1000 Genomes than in Diversity Outbred mice
I queried the 1000 Genomes Phase III VCF files for all SNPs that intersect with coding regions and calculated the minor allele frequency. I imputed the Sanger SNPs onto DO samples that were genotyped on the MegaMUGA between generations 7 to 12 and intersected them with coding regions and calculated the minor allele frequency.

16. Only 10% of 1000 Genomes SNPs have a minor allele frequency (MAF) > 5%. 98% of DO SNPs have an MAF > 5%.
9.6%
MAF > 5%
MAF < 5%
98.6%

17. The situation does not improve with coding SNPs.
7.1%
MAF > 5%
MAF < 5%
98.6%

18. Low minor allele frequency decreases power
MAF = 5%

19. Mice have more SNPs with MAF > 5%
1000 Genomes: 2,504 samples
84,801,880 SNPs
Diversity Outbred: 2,936 samples
40,425,646 SNPs

20. Simple Mapping Example: Coat Color
Coat color in ~850 DO mice recorded as:
Albino
Not-Albino
Mapped Albino as a binary trait.
Map using two methods:
Linkage mapping using haplotypes.
Association mapping using imputed SNPs.
Albino is homozygous recessive.

21. Albino coat color is a Mendelian trait regulated by a gene on Chr 7

22. We can distinguish the effects of each founder allele in the DO
Albino allele
A/J
NOD/ShiLtJ
Chr 7 (Mb)

23. Sanger All eight DO founders have been fully sequenced
Sanger Mouse Genomes Project:

24. Sanger All eight DO founders have been fully sequenced

25. We can impute the founder SNPs onto each DO genome

26. Impute founder SNPs and perform association mapping
A/J
NOD/ShiLtJ
Tyrc

27. Mapping Example: Benzene Genotoxicity

28. Benzene Inhalation Study
Day 0 7 14 21 28 1 2
Total 75
150
300
600
0 ppm
1 ppm
10 ppm
100 ppm
6 hrs per day
5 days per week

29. Benzene Study Endpoints
Pre- and post-exposure blood
Post-exposure bone marrow
Proportion of micronucleated reticulocytes (MN-RET)
Measure of chromosomal damage
French, et.al., Environmental Health Perspectives, 2015
PubMed Link

30. Bone Marrow MN-RET
DNA
Damage
French et.al., EHP, 2015

31. Linkage Mapping Model Map with 150 samples from 100 ppm dose.
yi = phenotype for sample i.
bj = founder allele effect for founder j.
hij = founder haplotype contribution for sample i for founder j.
li = correction for population structure.

32. QTL Plot of Bone Marrow MN-RET
French et.al., EHP, 2015

33. The CAST allele is protective
DNA
Damage
Chr 10
French et.al., EHP, 2015

34. The CAST allele is dominant
Prop. MN-RET
Genotype
French et.al., EHP, 2015

35. We can impute the founder SNPs onto each DO genome

36. Association Mapping Model
Map with 150 samples from 100 ppm dose.
yi = phenotype for sample i.
bj = SNP allele effect for founder j.
aij = contribution of allele j for sample i.
li = correction for population structure.

37. Sult3a1 and Gm4794 are sulfotransferases

38. Sult3a1 and Gm4794 have high expression in CAST livers

39. CAST/EiJ has a copy number gain of sulfotransferases
Gm4794
Sult3a1

40. Hypothesis Sulphate conjugation leads to excretion
Metabolite produces ROS

41. Hypothesis Bone Marrow Liver Bone Marrow Liver More excretion
Mice with other allele at Chr 10 QTL
Active metabolites
Bone Marrow
Liver
DNA Damage
Less excretion
Mice with CAST/EiJ allele at Chr 10 QTL
Active metabolites
Bone Marrow
Liver
Less DNA Damage
More excretion

42. Summary
Multi-parent, multi-generation crosses offer high genetic diversity and fine recombination block structure.
Increased complexity requires specialized methods for haplotype reconstruction and mapping.
QTL confidence intervals are several Mb.
Founder sequences can help to identify causal variants.

43. Acknowledgements UNC-CH Fernando Pardo-Manuel de Villena Daniel Pomp
The Jackson Laboratory Gary Churchill Neal Goodwin Elissa Chesler Karen Svenson Steven Munger Narayanan Raghupathy Al Simons Kwangbom Choi Petr Simecek Ivan Dotu Mark P. Keller Jeff Chuang Alan Attie Anne Greenlee Joel Graber Steve Ciciotte Lisa Somes, Doug Hinerfeld / Sandy Daigle/ Sonya Kamdar
UNC-CH
Fernando Pardo-Manuel de Villena
Daniel Pomp
Leonard McMillan
Catherine Walsh
Cheng-ping Fu
Univ. of Wisconsin
Karl Broman
Univ. of Chicago
Abraham Palmer
Riyan Cheng
NIEHS
John E. French
Kristine Witt
Daniel Morgan
Grace Kissling
Keith Shockley
ILS, Inc.
Kim Shepard
Leslie Recio
Alion
Herman Price