Download Multimodal Approaches to QTL Analysis.

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
Phenotype to Gene
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.
100
90
80
NR
60
50
50
40
40
30
Percent
Percent Responding
70
30
20
NNR
NR
NNR
20
10
10
0
0
Albino
Piebald
0
3
5
7
9
11
14
16
Generation
0
4
8
12
Generation
16
20
24
20
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
Selective
Breeding
Phenotype to Gene
Selective
Breeding
Basic
Genetics
Phenotype to Gene
Haloperidol ED50 (mg/kg)
+/- S.D.
4
3
2
1
0
Isogenic Generations
Segregating Generations
B6
BcB6
F1
BcD2
F2
0%
50%
Gene Dosage (% D2 alleles)
D2
100%
Activity - cm/5 min
6000
Basal Activity
5000
C57BL/6J
DBA/2J
F1
F2
4000
3000
2000
1000
0
5
10
Time
15
20
Activity - cm/5 min
3000
Ethanol-Induced
Activity
2500
C57BL/6J
DBA/2J
F1
F2
2000
1500
1000
500
0
5
10
Time
15
20
Scatterplot: ETHANOL1 vs. ETHANOL1 (Casewise MD deletion)
Scatterplot: SALINE1T vs. SALINE1T (Casewise MD deletion)
ETHANOL1 = 2621.2 + .26595 * ETHANOL1
Correlation: r = .27391
SALINE1T = 3013.6 + .59582 * SALINE1T
Correlation: r = .53850
25000
24000
22000
20000
20000
18000
15000
16000
14000
SALINE1T
ETHANOL1
10000
5000
12000
10000
8000
0
6000
-5000
4000
2000
-10000
0
-15000
-20000 -15000 -10000
-5000
0
5000
ETHANOL1
10000
15000
20000
25000
30000
95% confidence
-2000
-2000
0
2000
4000
6000
8000
10000 12000 14000 16000 18000 20000
SALINE1T
95% confidence
Scatterplot: SALINE1T vs. SALINE1T (Casewise MD deletion)
Test - Retest - Total Basal Activity - C57BL/6 x LP
SALINE1T = 3013.6 + .59582 * SALINE1T
Correlation: r = .53850
Var9 = 442.20 + .68757 * Var8
Correlation: r = .72346
24000
26000
22000
24000
20000
22000
18000
20000
18000
16000
16000
14000
12000
Var9
SALINE1T
14000
10000
12000
10000
8000
8000
6000
6000
4000
4000
2000
2000
0
0
-2000
-2000
0
2000
4000
6000
8000
10000 12000 14000 16000 18000
SALINE1T
20000
95% confidence
-2000
-2000
0
2000
4000
6000
8000 10000 12000 14000 16000 18000 20000 22000
Var8
95% confidence
Selective
Breeding
Basic
Genetics
Phenotype to Gene
Recombinant
Inbred
Strains
Key Publications for QTL Analysis in 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.
19
11
28
BXD Strain
20
12
6
25
B6
32
16
18
23
29
7.90
6.67
5.89
4.43
4.18
3.81
3.80
3.62
3.25
2.94
2.80
2.73
2.47
2.39
2.31
1.73
1.53
1.48
1.35
1.23
1.17
1.17
C57L
14
LP
9
129
13
CBA
8
P
27
B6
5
A/He
15
A
1
C3H
21
SJL
31
CE
24
PL
30
D2
D2 22
0.98
6
0.96
4
AKR
0
8
0.93
6
0.61
7
0.55
8
BALB/c
0.4
9
10
Standard
Inbred Strains
4
2
5
3
2
1
2
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
11q23-q24
15
20
25
11q23-q24
11q23-q24
15q23-q24
15q22-q24
Drd2
(0.55)
30
35
Fli-1 (0.33)
Lap-1 (0.27)
Xmv-16 (0.40)
T3d (0.40)
Apoa-1 (0.40)
Ncam, D9Mit22 (0.55)
D9Mit4, D9Byu1b (0.54)
Xmv-15 (0.38)
40
d
D9Mit8 (0.04)
45
Htr1b
50
Chromosome 9
Key Publications for QTL Analysis in 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.
Key Publications for QTL Analysis in 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
Selective
Breeding
Basic
Genetics
Intercross
Mapping
Phenotype to Gene
Recombinant
Inbred
Strains
Phenotype = Haloperidol-Induced Catalepsy
Cpu3
El1
Acra3
Htr1b
Myo5a
El4
Grik4
Scn2b
Ncam
Drd2
Acat1
Acra5
Acra5
Penk2
Gria4
D9Mit212
D9Mit111
D9Mit74
D9Mit144
D9Mit22
D9Mit21
D9Mit206
D9Mit188
D9Mit90
2
LOD
Phenotype = Ethanol-Induced Locomotor Activation
6
1
D2MIT229
D2MIT458
D2MIT241
D2MIT94
D2MIT420
D2MIT102
D2MIT491
D2MIT493
D2MIT412
D2MIT282
.
. . . . . ..
..
D2MIT521
.. .
I
Free
Dominant
Recessive
Additive
10 cM
0
D2MIT464
D2MIT80
Ntsr
Lorr2
Snap25
Adra2b
Plcb2
Kcna1-rs2
Gad1
Scn1a
Alcp1q
N = 600
Chromosome 2
5
4
3
Ethanol-Induced Locomotor Activation
30
F-Value
25
B6D2 Intercross
0 - 5 Min
5 - 10 Min
10 - 15 Min
15 - 20 Min
20
15
10
5
0
0
N = 1820
20
40
cM
60
Chromosome 2
80
100
50
Comparison of IM and CIM Techniques
40
LOD
30
20
10
0
0
1
Relative Chromosome Length
10
14
8
Additive
Free
Additive
12
6
10
LOD
LOD
Ethanol Response 10-15 Min (CIM)
16
Ethanol Response 0-5 Min (CIM)
4
2
Free
8
6
4
Dominant
Dominant
2
0
0
0
20
40
60
80
cM - Chromosome 2
100
120
0
Ethanol Response 5-10 Min (CIM)
10
40
60
80
cM - Chromosome 2
100
120
Ethanol Response 15-20 Min (CIM)
14
12
8
Additive
Free
10
6
8
LOD
LOD
20
4
Free
Additive
6
4
2
Dominant
2
0
Dominant
0
0
20
40
60
80
cM - Chromosome 2
100
120
0
20
40
60
80
cM - Chromosome 2
100
120
Evidence for epistasis – basal locomotor activity
D1Mit365
-------
B6:B6
B6:D2
D2:D2
--:--
D8Mit236
--------------------------------------------------------------------------------------------:--
B6:B6
B6:D2
D2:D2
2660 + 88
2180 + 95***
1790 + 58****
2250 + 166***
2380 + 113
1870 + 172
2300 + 115
2650 + 238
2090 + 88
1910 + 106
2080 + 113
3080 + 198
2090 + 173
1600 + 280
2260 + 98
Koyner et al. 2000
Selective
Breeding
Fine
Mapping
Basic
Genetics
Phenotype to Gene
Recombinant
Inbred
Strains
Intercross
Mapping
Key Publications for QTL Analysis in 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.
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
Ethanol-Induced Locomotor Activation
30
F-Value
25
B6D2 Intercross
0 - 5 Min
5 - 10 Min
10 - 15 Min
15 - 20 Min
20
15
10
5
0
0
20
40
cM
60
80
100
N = 1820
30
0 - 5 Min
5 - 10 Min
10 - 15 Min
15 - 20 Min
F-Value
20
10
0
-10
HS Population
-20
45
50
55
60
cM
N = 550 @ G32 Chromosome 2
65
70
75
Mechanism
& Circuit
Basic
Genetics
Selective
Breeding
Phenotype to Gene
Recombinant
Inbred
Strains
Intercross
Mapping
Fine
Mapping
Fos Positive Cells / Section
80
Central Amygdaloid
Nucleus
DBA
60
C57
40
20
0
0.25
0.5
1
Ethanol - g/kg
2
4
GABA
(Peptide)
DA, NE
5-HT, ACh
BST
4
Glu
(Peptide)
GABA
(Peptide)
GABA
2
5
3
CeL
1
CeM
Glu
(Peptide)
Ins Cx, , PB
Motor Response
Multiple Cross Mapping (MCM): A New
Recipe for QTL Measurement and Gene
Detection
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.
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.
QTL Maps - Ethanol Response
6
5
C57BL/6 x DBA/2
C57BL/6 x LP
C57BL/6 x BALB/c
-Log P
4
3
2
1
0
0
20
40
60
80
100
120
60
80
100
120
6
5
C57BL/6 x DBA/2
BALB/c x DBA/2
DBA/2 x LP
BALB/c x LP
-Log P
4
3
2
1
0
0
20
40
cM - Chromosome 1
0.35
Fraction of Total Markers
Fraction of Total Markers
0.14
0.12
0.10
0.08
C57BL/6 vs DBA/2
Distribution of Marker
Set Based On
QTL Criteria
0.30
0.25
0.20
Polymorphic
Not Polymorphic
0.15
0.10
0.05
0.00
0
20
40
60
80
100
120
Chromosome 1 - cM
0.06
0.04
0.02
0.00
0
20
40
60
80
Chromosome 1 - cM
100
120
Table 1: Analysis of Chromosome 1 QTL for Ethanol Response in HS Mice
11
Markers (Catalog Position-cM)
Calculated Interval - cM
- Log P
0
1
D1Mit 103(73) - D1Mit423(73)
D1Mit423(73) - D1Mit 100(71.5)
64.100 - 64.730
64.730 - 64.760
2.46
2.68
2
D1Mit 100(71.5) - D1Mit496(70)
64.760 - 64.840
3.18
3
4
D1Mit496(70) - D1Mit264(71.5)
D1Mit264(71.5) - D1Mit289(74.3)
64.860 - 64.960
64.960 - 65.170
3.30
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
8
D1Mit268(83.4) - D1Mit452(86.6)
D1Mit452(86.6) - D1Mit16(87.2)
71.980 - 73.880
73.880 - 75.100
10.8
2.72
9
D1Mit16(87.2) - D1Mit370(87.9)
75.100 - 77.280
2.99
10
11
D1Mit370(87.9) - D1Mit110(88.1)
D1Mit110(88.1) - D1Mit15(87.9)
77.280 - 77.550
77.550 - 77.960
2.99
3.51
12
D1Mit15(87.9) - D1Mit270(92.3)
77.960 - 80.270
1.91
13
14
D1Mit270(92.3) - D1Mit36(92.3)
D1Mit36(92.3) - D1Mit113(93.3)
80.270 - 80.800
80.800 - 81.400
0.91
3.06
15
D1Mit113(93.3) - D1Mit402(92.3)
81.400 - 81.900
1.95
16
17
D1Mit402(92.3) - D1Mit149(94.2)
D1Mit149(94.2) - D1Mit354(95.8)
81.900 - 82.500
82.500 - 82.600
1.26
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
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)
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
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
Chromosome 2
Gene
cM
F-value
glutamate receptor, ionotropic, NMDA1 (zeta 1)
prostaglandin D2 synthase (21 kDa, brain)
prostaglandin D2 synthase (21 kDa, brain)
prostaglandin D2 synthase (21 kDa, brain)
heat shock 70kD protein 5 (glucose-regulated protein, 78kD)
pre B-cell leukemia transcription factor 3
gelsolin
tumor necrosis factor induced protein 6
neurogenic differentiation 1
CD59a antigen
reticulocalbin
secretory granule neuroendocrine protein 1, 7B2 protein
maternal inhibition of differentiation
beta-2 microglobulin
protein tyrosine phosphata
cystatin C
inhibitor of DNA binding 1
neuronatin
protein kinase inhibitor, gamma
protective protein for beta-galactosidase
SRY-box containing gene 18
potassium voltage-gated channel, subfamily Q, member 2
myelin transcription factor 1
sialyltransferase 7
transcription termination factor 1
c-mer proto-oncogene
fibroblast growth factor 7
isovaleryl coenzyme A dehydrogenase
12
13
13
13
22
22
25
30
46
55
58
64
68
69
73
84
84
88
94
96
96
104
106
7
12
8
7
13
8
26
19
6
50
15
129
10
27
6
21
19
55
67
9
7
21
10
7
7
9
25
43
QTL Maps
Ethanol Response
600
Diff. Score
14
12
C57BL/6 x DBA/2
BALB/c x DBA/2
LP x DBA/2
F value
10
Reticulocalbin
500
400
300
200
100
0
8
C57BL/6 DBA/2
BALB/c
LP/J
6
4
2
2000
Sgne1
0
20
40
60
80
Diff. Score
0
100
14
C57Bl/6 x DBA/2
C57BL/6 x BALB/c
LP x BALB/c
C57Bl/6 x LP
12
8
1000
500
0
C57BL/6 DBA/2
BALB/c
LP/J
6
4
2
120
0
100 Microglobulin
0
20
40
60
80
Chromosome 2 - cM
100
120
Diff. Score
F Value
10
1500
Beta-2
80
60
40
20
0
C57BL/6 DBA/2
BALB/c
LP/J
D2 = C = LP Cas1
Catalase Activity
QTL Maps
Ethanol Response
14
12
C57BL/6 x DBA/2
BALB/c x DBA/2
LP x DBA/2
F value
10
8
6
4
2
140
130
120
110
100
90
80
70
60
RI-#32
RI-#11
RI-#28
D2
RI-#16
RI-#6
RI-#1
B6
B6
D2
Allele
0
0
20
40
60
80
100
14
C57Bl/6 x DBA/2
C57BL/6 x BALB/c
LP x BALB/c
C57Bl/6 x LP
12
F Value
10
8
Bdnf
6
4
2
0
0
20
40
60
80
Chromosome 2 - cM
100
120
B6  Leu
D2  Met
C  Met
LP  Leu
QTL Maps - Basal Actvity
25
B6 vs D2
B6 vs LP
B6 vs C
F-value
20
15
10
5
0
0
20
40
60
80
100
120
40
60
80
100
120
25
B6 vs D2
LP vs D2
Lp vs C
C vs D2
F value
20
15
10
5
0
0
20
cM - chromosome 1
Chromosome 1
Gene
Eph receptor A4
myosin Ib
acetyl-Coenzyme A dehydrogenase, long-chain
isocitrate dehydrogenase 1 (NADP+), soluble
insulin-like growth factor binding protein 5
insulin-like growth factor binding protein 2
peptidylglycine alpha-amidating monooxygenase
troponin T2, cardiac
astrotactin 1
POU domain, class 2, transcription factor 1
pre B-cell leukemia transcription factor 1
peroxisomal farnesylated protein
potassium inwardly-rectifying channel, subfamily J, member 9
ATPase, Na+/K+ transporting, alpha 2 polypeptide
LanC (bacterial lantibiotic synthetase component C)-like
growth arrest specific 5
transcription elongation factor A (SII) 1
kinesin heavy chain member 1A
expressed sequence C77892
cM
F-value
7
25
27
30
36
36
58
60
85
87
88
93
94
94
6
6
7
6
23
8
61
11
7
17
6
13
172
7
104
61
55
22
9
Diff. Score
QTL Maps - Basal Actvity
25
B6 vs D2
B6 vs LP
B6 vs C
F-value
20
15
180
160
140
120
100
80
60
40
20
0
Pxf
C57BL/6
DBA/2
BALB/c
LP
C57BL/6
DBA/2
BALB/c
LP
10
GIRK3
5
600
0
20
40
60
80
100
120
25
B6 vs D2
LP vs D2
Lp vs C
C vs D2
15
400
300
C57BL/6
DBA/2
BALB/c
LP
200
100
0
10
5
0
0
20
40
60
80
cM - chromosome 1
100
120
Diff. Score
F value
20
Diff. Score
500
0
1800
1600
1400
1200
1000
800
600
400
200
0
Atp1a2
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
…………
GABA Related Genes
Gene
Chr
cM
Gabra1
Gabra2
Gabra6
Gabrb1
Gabrb2
Gabrb3
Gabrg1
Gabrg2
Gabrg3
Gabt1
Gad1
Gad2
11
5
11
5
11*
7
5
11
7
3
2
2
19cM
40cM
23cM
40cM
*25cM
29cM
40cM
19cM
28cM
50cM
43cM
9cM
Viaat
2
80cM
F-value P-value
C57BL/6J
DBA/2J
LP/J
BALB/cJ
16.7756
17.5145
13.5512
13.5435
10.0278
1.8440
5.8868
3.0945
1.3917
0.4664
3.0010
2.1515
0.000011
0.000008
0.000047
0.000047
0.000304
0.171715
0.004744
0.050183
0.274346
0.708949
0.054809
0.125644
98
0.000000
Mean + SD (N=6/strain)
526+44
298 + 15
505 + 59
497 + 103
51 + 18
140 + 29
125 + 26
132 + 23
672 + 81
848 + 89
901 + 57
922 + 71
965 + 85
1085 + 101 769 + 78
969 + 83
34 + 14
2+6
44 + 17
46 + 21
69 + 23
83 + 22
99 + 28
86 + 17
40 + 8
51 + 9
64 + 14
57 + 10
801 + 45
881 + 72
902 + 77
888 + 54
71 + 25
87 + 17
80 + 11
91 + 15
1064 + 90
1105 + 76
1110 + 65 1092 + 58
1900 + 116 1973 + 123 2055 + 123 2082 + 93
61 + 16
75 + 13
84 + 19
89 + 29
1434 + 70
748 + 109 1494 + 88 1456 + 80
Comparison of GABA Receptor a-Subunit Gene
Expression in Whole Brain, the Dorsomedial Striatum
and the Central Extended Amygdala.
Gene
Chr
Gabra1
11
DBA/2J
LP/J
Mean + SD (N=3-6/strain)
19cM
WB
526 + 44
298 + 15
505 + 59
Striatum (DM) 272 + 106 143 + 41
195 + 55
CeA-BSTLP 475 + 35
220 + 19
318 + 29
Gabra2
5
40cM
Gabra3
X
Gabra6
11
A = absent
cM
23cM
Region
C57BL/6J
BALB/cJ
497 + 103
258 + 23
386 + 56
WB
Striatum (DM)
CeA-BSTLP
51 + 18
57 + 17
108 + 10
140 + 29
186 + 21
211 + 44
125 + 26
168 + 32
217 + 24
132 + 23
196 + 15
213 + 59
WB
Striatum (DM)
CeA-BSTLP
A
A
40 + 17
A
A
25 + 8
A
A
33 + 13
A
A
23 + 21
WB
Striatum (DM)
CeA-BSTLP
672 + 81
10 + 4
A
848 + 89
8+6
A
901 + 57
20 + 18
A
922 + 71
13 + 6
A
QTLs and GABA Related Gene Expression
• Chr 11 (a1, a6, b2, g2) – Acute pentobarbital
withdrawal, acute alcohol withdrawal, forced
swim test, light-dark latency, tail suspension
• Chr 5 (a2,b1,g1) – kianic acid induced seizures,
PTZ induced seizures, alcohol hypothermic
sensitivity and tolerance, (elevated plus maze
behavior)
• Chr 2 (Viaat) – acute locomotor response to
ethanol and chlordiazepoxide
QTL Maps - Ethanol Response
6
5
C57BL/6 x DBA/2
C57BL/6 x LP
C57BL/6 x BALB/c
-Log P
4
3
2
1
0
0
20
40
60
80
100
120
60
80
100
120
6
5
C57BL/6 x DBA/2
BALB/c x DBA/2
DBA/2 x LP
BALB/c x LP
-Log P
4
3
2
1
0
Ethanol-Induced Response cm/20 min
6000
5000
Genotype vs Response at D1Mit150
Four-way Cross
4000
3000
2000
1000
0
B6:B6 C:C B6:D2 B6:LP D2:LP
D2:D2 LP:LP B6:C
D2:C C:LP
0
20
40
cM - Chromosome 1
Mechanism
& Circuit
Basic
Genetics
Selective
Breeding
Multiple
Cross
Mapping
Phenotype to Gene
Recombinant
Inbred
Strains
Functional
Genomics
Intercross
Mapping
Fine
Mapping
Complex Trait
Probable Gene
Intestinal tumor resistance
Pla2g2a
Mom1 [mice]
Type II diabetes-insulin
resistance [rats]
Cd36
Type I diabetes-
Idd3
islet degeneration [mice]
Allergen-induced airway
hyperresponsivenessasthma [mice]
Susceptibility to lupus
[mice]
Cardiac ventricular
hypertrophy [rats]
Blood pressure
[rats]
Sweet taste preference
behavior [mice]
Cd5
Ifi202
Nppa
Cyp11b1
Tas1r3
Reference
Cormier et al., 1997
Nature Genet 17:88
Aitman et al., 1999; Pravenac
et al., 2001, Nature
Genet 16:197 & 27:156
Lyons et al., 2000
Genome Res 10:446
Karp et al., 2000
Nature Immun 1:221
Rozzo et al., 2001
Immunity 15:435
Deschepper et al., 2001
Circul Res 88:223
Cicila et al., 2001
Genomics 72:51
Max et al., 2001
Nature Genet 28:58
JKB, Dec 2001
Complex Trait Probable Gene Method of gene ID
Intestinal tumor resistance
Pla2g2a
Mom-1 [mice]
Type II diabetes-insulin
resistance [rats]
Cd36
Type I diabetes-
Idd3/Il2
islet degeneration [mice]
Allergen-induced airway
hyperresponsivenessasthma [mice]
Susceptibility to lupus
[mice]
Cardiac ventricular
hypertrophy [rats]
Blood pressure
[rats]
Sweet taste preference
behavior [mice]
Cd5
Ifi202
Nppa
Cyp11b1
Tas1r3
High res mapping/Transgenic
rescue
Microarray analysis in congenic
vs background strain/
transgenic rescue
Mapping to 780 kb/
functional testing
Microarray analysis in congenic
vs background strain
Microarray analysis in congenic
vs background strain/functional
Promotor SNP/functional
testing/in vitro expression
Mapping to 220kb/SNPs/
functional testing
Taste cell specific expression/
glycosylation site SNP/ functional
testing.
JKB, Dec. 2001
25
Number of QTLs
20
15
Distribution of Some Murine
Behavioral QTLs - LOD > 3
Total Number of
QTLs = 124
10
Aggression
Circadian Rhythm
Drug Preference
Drug Response
Emotion
Learning
Motor Activity
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
Chromosome
Acknowledgements
Stony Brook
OHSU
Denver
Jim McCaughran
Laura Cipp
Kristin Demarest
Jay Koyner
Stephen Kanes
Nilay Patel
Erik Rasmussen
Barbara Hitzemann
Yifang Qian
Peter Thanos
Adena Svingos
Barry Malmanger
Shannon Coulombe
Staci Cooper
Cheryl Reed
Barbara Hitzemann
Maureen Lawler
Kristin Hitzemann
Tarra Gupta
Ronnie Dhamer
Jim Sikela
Yan Xu
Kari Buck
Brooks Rademacher
Oxford, UK
Jonathan Flint
Chris Talbot