Diversity Outbred Mice Daniel Gatti, Ph.D dan.gatti@jax.org May 2016 International Behavioural and Neural Genetics Society

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”

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

Genetically diverse mouse models may improve translational relevance

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

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

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

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

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

Collaborative Cross mice Available from Univ. of North Carolina. http://csbio.unc.edu/CCstatus/index.py ~ 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

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

Diversity Outbred mice Available from The Jackson Laboratory. http://jaxmice.jax.org/strain/009376.html 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.

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

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

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.

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%

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

Low minor allele frequency decreases power MAF = 5%

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

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.

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

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

Sanger All eight DO founders have been fully sequenced Sanger Mouse Genomes Project: http://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1505

Sanger All eight DO founders have been fully sequenced

We can impute the founder SNPs onto each DO genome

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

Mapping Example: Benzene Genotoxicity

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

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

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

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.

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

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

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

We can impute the founder SNPs onto each DO genome

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.

Sult3a1 and Gm4794 are sulfotransferases

Sult3a1 and Gm4794 have high expression in CAST livers http://cgd.jax.org/gem/strainsurvey26/v1

CAST/EiJ has a copy number gain of sulfotransferases http://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1303 Gm4794 Sult3a1 http://ensembl.org

Hypothesis Sulphate conjugation leads to excretion Metabolite produces ROS http://www.crios.be/Benzene/

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

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.

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