Robert Hitzemann, Ph.D. Department of Behavioral Neuroscience Oregon Health & Sciences University

1. Multimodal Complex Trait Analysis: Combined Use of F2, HS, RI, SNPs, SSLP Haplotypes and Arrays to Fine-Map Behavioral Traits" Robert Hitzemann, Ph.D. Department of Behavioral Neuroscience Oregon Health & Sciences University Portland, OR

2. Phenotype to Gene

3. The Beginning… Schueler HE, Hitzemann RJ, Harris RA, Kreishman GP (1989) Ethanol-induced differential disordering of synaptic plasma membranes from mice selected for genetic differences in ethanol intoxication. Prog Clin Bio Res 292: 425-434.

4. 100 90 80 NR 70 60 50 50 Percent Responding 40 40 Albino 30 Percent Piebald NNR 30 20 NR NNR 20 10 10 0 0 3 5 7 9 11 14 16 20 Generation 0 0 4 8 12 16 20 24 Generation

5. Selective Breeding as a Mode of Contemporary QTL Analysis. • Strong evidence that the phenotype of interest has a measurable heritability • Provides a mechanism for detecting correlated traits and thus potential candidate genes • Both short and long term selective breeding provide instruments for confirming QTLs • Selectively bred animals may prove useful for the fine mapping of QTLs

6. Phenotype to Gene Selective Breeding

7. Phenotype to Gene Selective Breeding Basic Genetics

8. 4 3 2 Haloperidol ED50 (mg/kg) +/- S.D. 1 Isogenic Generations Segregating Generations 0 B6 BcB6 F1 BcD2 D2 F2 0% 50% 100% Gene Dosage (% D2 alleles)

12. Phenotype to Gene Selective Breeding Basic Genetics Recombinant Inbred Strains

13. Key Publications for QTL Analysisin Mice. Plomin R. McClearn GE. Gora-Maslak G. Neiderhiser JM. Use of recombinant inbred strains to detect quantitative trait loci associated with behavior. Behavior Genetics. 21(2):99-116, 1991 Mar.

14. 9 8 10 Standard 8 7 Inbred Strains 6 4 6 2 5 A P D2 PL Haloperidol - ED 50 mg/kg B6 CE LP SJL 129 AKR C3H CBA A/He C57L BALB/c 4 3 2 1 0.96 0.98 1.17 1.17 1.23 1.35 1.48 1.53 1.73 2.39 2.47 2.73 2.80 2.94 3.25 3.62 3.80 3.81 4.18 5.89 6.67 7.90 0.93 2.31 4.43 0.55 0.61 0.4 0 D2 22 31 1 5 8 9 19 28 12 25 32 18 29 30 24 21 15 27 13 14 11 20 6 B6 16 23 2 BXDStrain

15. Conditional QTLs (p < 0.01) Obtained from the Analysis of the RI Strain Means • D2Mit238, D2Ncvs31, D2Mit7 (27-30 cM, r = -.54) • D4Mit17, b(Trp1), Ifa (29-42 cM, r = -.62) • D6Mit16, D6Mit9, Tgfa(26-33 cM, r = -.72) • D9Mit4, Ncam, Drd2, D9Mit21 (28-32 cM, r = 0.56) • D15Mit1 (41-48 cM, r = -.65) • D16Mit131 (10 cM, r = .52) 0 5 15 11q23-q24 Fli-1 (0.33) 20 Lap-1 (0.27) Xmv-16 (0.40) 25 T3d (0.40) 11q23-q24 Apoa-1 (0.40) Drd2 11q23-q24 Ncam, D9Mit22 (0.55) (0.55) D9Mit4, D9Byu1b (0.54) 15q23-q24 30 15q22-q24 35 Xmv-15 (0.38) 40 d D9Mit8 (0.04) 45 Htr1b 50 Chromosome 9

16. Key Publications for QTL Analysisin Mice. Plomin R, McClearn GE, Gora-Maslak G, Neiderhiser JM (1991) Use of recombinant inbred strains to detect quantitative trait loci associated with behavior. Behavior Genetics. 21(2):99-116. Belknap JK, Mitchell SR, Crabbe JC (1996) Type I and II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred mouse strains: Computer simulation and empirical results. Behavior Genetics 26:581-592.

17. Key Publications for QTL Analysisin Mice. Dietrich W, Katz H, Lincoln SE, Shin HS, Friedman J, Dracopoli NC, Lander ES (1996) A genetic map of the mouse suitable for typing intra-specific crosses.Genetics. 131(2):423-47. Flint J, Corley R, DeFreis JC, Fulker DW, Gray JA, Miller S, Collins AC (1995) A simple genetic basis for a complex psychological trait in laboratory mice. Science 269: 1432-35

18. Phenotype to Gene Selective Breeding Basic Genetics Intercross Mapping Recombinant Inbred Strains

19. 6 5 4 3 2 1 0 Chromosome 9 LOD Score D9Mit206 D9Mit188 D9Mit212 D9Mit111 D9Mit144 D9Mit90 D9Mit74 D9Mit21 D9Mit22 70 60 50 40 30 0 20 10 cM Myo5a Penk2 Scn2b Acra3 Acra5 Acra5 Ncam Gria4 Acat1 Cpu3 Htr1b Grik4 Drd2 El4 El1 Phenotype = Haloperidol-Induced Catalepsy

20. Phenotype = Ethanol-Induced Locomotor Activation 6 5 Chromosome 2 4 3 LOD 2 1 Free Dominant Recessive 0 Additive 10 cM . . . . . . . . . . . . . I D2MIT94 D2MIT229 D2MIT80 D2MIT412 D2MIT241 D2MIT282 D2MIT458 D2MIT464 D2MIT420 D2MIT102 D2MIT491 D2MIT493 D2MIT521 Ntsr Lorr2 Gad1 Plcb2 Adra2b Scn1a N = 600 Snap25 Alcp1q Kcna1-rs2

21. Ethanol-Induced Locomotor Activation N = 1820 Chromosome 2

22. 50 Comparison of IM and CIM Techniques 40 30 20 LOD 10 0 0 1 Relative Chromosome Length

24. Evidence for epistasis – basal locomotor activity D8Mit236 D1Mit365 ------------------------------------------------------------------------------------------- ------- -- : -- B6:B6 B6:D2 D2:D2 B6:B6 2660 + 88 2250 + 166 *** 2650 + 238 3080 + 198 B6:D2 2180 + 95*** 2380 + 113 2090 + 88 2090 + 173 D2:D2 1790 + 58**** 1870 + 172 1910 + 106 1600 + 280 -- : -- 2300 + 115 2080 + 113 2260 + 98 Koyner et al. 2000

25. Phenotype to Gene Selective Breeding Fine Mapping Basic Genetics Intercross Mapping Recombinant Inbred Strains

26. Key Publications for QTL Analysisin Mice. Darvasi A (1998) Experimental strategies for the genetic dissection of complex traits in animal models. Nature Genetics 18: 19-24. Talbot CJ, Nicod A, Cherny SS, Fulker DW, Collins AC, Flint J (1999) High resolution mapping of quantitative trait loci in outbred mice. Nature Genetics 21: 305-308. Threadgill DW, Hunter KW, Williams RW (2002) Genetic dissection of complex and quantitative traits: from fantasy to reality via a community effort. Mammalian Genome 13: 175-8.

27. Strategies for Fine Mapping (< 1 cM) QTLs. • Interval specific congenic strains • Advanced F2 intercrosses • Heterogeneous stock (HS) > G30 • Large (~1,000 strains) recombinant inbred panels – also includes the use of RIX animals • The integration of functional genomics and QTL analysis

30. Ethanol-Induced Locomotor Activation N = 1820 Chromosome 2 N = 550 @ G32

31. Phenotype to Gene Mechanism & Circuit Basic Genetics Selective Breeding Fine Mapping Recombinant Inbred Strains Intercross Mapping

32. Central Amygdaloid • Nucleus 80 DBA 60 C57 Fos Positive Cells / Section 40 20 1 2 0.5 4 0.25 0 Ethanol - g/kg

33. GABA DA, NE (Peptide) 5-HT, ACh BST 4 Glu GABA 5 GABA (Peptide) (Peptide) 2 3 CeM CeL Glu (Peptide) 1 Motor Response Ins Cx, , PB

34. Multiple Cross Mapping (MCM): A New Recipe for QTL Measurement and Gene Detection

35. Multiple Cross Mapping (MCM) • Builds from the observation that for open-field/basal activity, three different diallele crosses (B6xC, B6xA and B6xD2) appear to generate some similar QTLs, most notably on distal chromosome1 (Hitzemann et al. 2000). One interpretation of these data would be that the C, A and D2 strains have common allele(s) in the region of interest. • The more general interpretation is that polymorphic and non-polymorphic alleles are not randomly distributed and thus, provide a source of information. MCM can be used to “mine” this information to reduce the QTL interval. • MCM provides a mechanism to interrogate the data obtained from gene and protein array analysis.

36. Ingredients for MCM (v2.0) • Four inbred mouse strains – C57BL/6J (B6), DBA/2J (D2), BALB/cJ (C) and LP/J (LP). • The six F2 intercrosses that can be produced from these four strains. • A four-way cross of the strains – Heterogeneous Stock 4 (HS4) at G11. • An eight way HS at G44 containing the above four strains plus the AKR/J, A/J, C3H/HeJ and CBA/J strains (HS8). • Chromosome-gene expression maps for the inbred strains. • Dense microsatellite and SNP maps for the inbred strains. • Multiple phenotypes: basal activity, ethanol-induced activity, haloperidol-induced catalepsy, the acoustic startle response (ASR) and pre-pulse inhibition (PPI) of the ASR.

39. Table 1: Analysis of Chromosome 1 QTL for Ethanol Response in HS Mice 1 1 - Log P Markers (Catalog Position-cM) Calculated Interval - cM 0 D1Mit 103(73) - D1Mit423(73) 64.100 - 64.730 2.46 1 D1Mit423(73) - D1Mit 100(71.5) 64.730 - 64.760 2.68 2 D1Mit 100(71.5) - D1Mit496(70) 64.760 - 64.840 3.18 3 D1Mit496(70) - D1Mit264(71.5) 64.860 - 64.960 3.30 4 D1Mit264(71.5) - D1Mit289(74.3) 64.960 - 65.170 5.86 5 D1Mit289(74.3) - D1Mit425(81.6) 65.170 - 68.010 13.5 6 D1Mit425(81.6) - D1Mit268(83.4) 68.010 - 71.980 12.7 7 D1Mit268(83.4) - D1Mit452(86.6) 71.980 - 73.880 10.8 8 D1Mit452(86.6) - D1Mit16(87.2) 73.880 - 75.100 2.72 9 D1Mit16(87.2) - D1Mit370(87.9) 75.100 - 77.280 2.99 10 D1Mit370(87.9) - D1Mit110(88.1) 77.280 - 77.550 2.99 11 D1Mit110(88.1) - D1Mit15(87.9) 77.550 - 77.960 3.51 12 D1Mit15(87.9) - D1Mit270(92.3) 77.960 - 80.270 1.91 13 D1Mit270(92.3) - D1Mit36(92.3) 80.270 - 80.800 0.91 14 D1Mit36(92.3) - D1Mit113(93.3) 80.800 - 81.400 3.06 15 D1Mit113(93.3) - D1Mit402(92.3) 81.400 - 81.900 1.95 16 D1Mit402(92.3) - D1Mit149(94.2) 81.900 - 82.500 1.26 17 D1Mit149(94.2) - D1Mit354(95.8) 82.500 - 82.600 0.39 18 D1Mit354(95.8) - D1Mit456(95.8) 82.600 - 82.800 0.33 19 DiMit456(95.8) - D1Mit541(97.7) 82.800 - 83.500 0.41 20 D1Mit541(97.7) - D1Mit115(99.7) 83.500 - 84.340 2.71

40. The Problem. • “All genetic studies begin with variation because perturbations in biological processes reveal functionally important elements in the life history of an organism. Naturally occurring variation still drives many studies because complex genetic traits remain important in humans, model organisms and agriculturally important animal and plant species. The existing challenge in complex trait analysis is to distinguish the mutations responsible for trait variability from closely linked, selectively neutral polymorphisms.” • Nadeau and Dunn (1998)

41. Gene Expression Project • Affymetrix “A” chip & 16,000 gene cDNA arrays for some groups of interest • Whole brain data in C57BL/6, DBA/2, BALB/c, LP, A, AKR, C3H/He, CBA and 129/SV – N=6-8/strain • Data from extended amygdala (CeA+BSTLP) and dorsomedial striatum both at baseline and after acute haloperidol (1 mg/kg) or acute ethanol (1.5g/kg) treatment; data will be obtained in C57BL/6, DBA/2, BALB/c, LP, A and 129/SV strains – N=6-8/strain • Purpose: to construct chromosome expression maps for the various strains and to integrate expression maps with sequence maps • Samples from other brain regions have been collected e.g. VTA, SNc, Cb and are available for analysis

42. Results of the Gene Expression Project to Date • Whole brain data (Affymetrix) have been obtained from 4 strains (B6, D2, C and LP) – N = 6 • ~6000 transcripts were detected as present in one or more of the strains; the actual number of genes expressed will be substantially fewer given that some genes have multiple transcripts • ~ 1900 transcripts were detected as being differentially expressed among the four strains at p < 0.05 • ~ 400 transcripts were detected as being differentially expressed among the four strains at p < 0.000005

43. Chromosome 2

45. Bdnf Cas1 D2 = C = LP 140 B6  Leu D2  Met C  Met LP  Leu RI-#32 RI-#11 130 RI-#28 120 D2 Catalase Activity 110 RI-#16 100 90 80 RI-#6 RI-#1 70 B6 60 B6 D2 Allele

47. Chromosome 1

49. Polymorphisms Between the C57BL/6J and DBA/2J Strains in Kcnj9 (GIRK3) 1 gggaacctag ggtactgggg gagatggtgt cagggacatg gacgccaacc cccaagggtt 61 tctgctgctg gctactcttc tctccaggct ctgggaccct gaaagcctag gaaccgactc 121 tggccatcca tctctccggg aagattataa cccagagtgc ttctcagggg ggaagaattt 181 gaagcaaaac cagaccccgc aggatccccg ctgcggccgc catgcgccag gagaacgccg 241 ctttctctcc cgggtcggag gagccgccac gccgccgcgg tcgccagcgc tacgtggaga ……….. 481 gcgacctgga gcacctggag gacaccgcgt ggaccccgtg cgtcaacaac ctcaacggct 541 tcgtggccgc cttcctcttc tccatagaga cggagaccac carcggctat gggcaccgcg ……….. 781 gtctcagtt tcgcgtgggc gaccctgcgat cctcacacat cgtcgaggcc tccatccgag ……….. 1081gccaagctcg aagctcgtac ctggtggatg aagtgttgtg gggccaccgg ttcacatccg ……….. 1201aggtgcccac accctcgtcg agtgcttcggg aactggcaga agccgcggcc cgccttgatg …………

50. Gene Chr cM F-value P-value C57BL/6J DBA/2J LP/J BALB/cJ Gabra1 11 19cM 16.7756 0.000011 526 + 44 298 + 15 505 + 59 497 + 103 Gabra2 5 40cM 17.5145 0.000008 51 + 18 140 + 29 125 + 26 132 + 23 Gabra6 11 23cM 13.5512 0.000047 672 + 81 848 + 89 901 + 57 922 + 71 Gabrb1 5 40cM 13.5435 0.000047 965 + 85 1085 + 101 769 + 78 969 + 83 Gabrb2 11* *25cM 10.0278 0.000304 34 + 14 2 + 6 44 + 17 46 + 21 Gabrb3 7 29cM 1.8440 0.171715 69 + 23 83 + 22 99 + 28 86 + 17 Gabrg1 5 40cM 5.8868 0.004744 40 + 8 51 + 9 64 + 14 57 + 10 Gabrg2 11 19cM 3.0945 0.050183 801 + 45 881 + 72 902 + 77 888 + 54 Gabrg3 7 28cM 1.3917 0.274346 71 + 25 87 + 17 80 + 11 91 + 15 Gabt1 3 50cM 0.4664 0.708949 1064 + 90 1105 + 76 1110 + 65 1092 + 58 Gad1 2 43cM 3.0010 0.054809 1900 + 116 1973 + Gad2 2 9cM 2.1515 0.125644 61 + 16 75 + 13 84 + 19 89 + 29 GABA Related Genes Mean + SD (N=6/strain) 123 2055 + 123 2082 + 93 1434 + 70 748 + 109 1494 + 88 1456 + 80 Viaat 2 80cM 98 0.000000