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 plasmamembranes from mice selected for genetic differences in
ethanol intoxication. Prog Clin Bio Res 292: 425-434.
0 4 8 12 16
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90
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020 24
Generation
Per
cen
t R
esp
on
din
g
NR
NNR
0 3 5 7 9 11 14 16 20
50
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NR
AlbinoPiebald
Generation
Per
cent
NNR
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
Phenotype to Gene
SelectiveBreeding
Phenotype to Gene
Basic Genetics
Selective Breeding
Hal
op
erid
ol
ED
50 (
mg
/kg
)+
/- S
.D.
D2BcD2F1BcB6B6
4
3
2
1
0
F2
Gene Dosage (% D2 alleles)0% 50% 100%
Isogenic GenerationsSegregating Generations
Basal Activity
Time5 10 15 20
Act
ivit
y -
cm/5
min
0
1000
2000
3000
4000
5000
6000
C57BL/6JDBA/2JF1F2
Ethanol-InducedActivity
Time5 10 15 20
Act
ivit
y -
cm/5
min
0
500
1000
1500
2000
2500
3000
C57BL/6JDBA/2JF1F2
Scatterplot: ETHANOL1 vs. ETHANOL1 (Casewise MD deletion)
ETHANOL1 = 2621.2 + .26595 * ETHANOL1Correlation: r = .27391
-20000 -15000 -10000 -5000 0 5000 10000 15000 20000 25000 30000
ETHANOL1
-15000
-10000
-5000
0
5000
10000
15000
20000
25000
ETHA
NOL1
95% confidence
Scatterplot: SALINE1T vs. SALINE1T (Casewise MD deletion)
SALINE1T = 3013.6 + .59582 * SALINE1TCorrelation: r = .53850
-2000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
SALINE1T
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
SALI
NE1T
95% confidence
Scatterplot: SALINE1T vs. SALINE1T (Casewise MD deletion)
SALINE1T = 3013.6 + .59582 * SALINE1TCorrelation: r = .53850
-2000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
SALINE1T
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
SA
LIN
E1T
95% confidence
Test - Retest - Total Basal Activity - C57BL/6 x LP
Var9 = 442.20 + .68757 * Var8Correlation: r = .72346
-2000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000
Var8
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
Var9
95% confidence
Phenotype to Gene
Basic Genetics
Selective Breeding
Recombinant
InbredStrains
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.
Key Publications for QTL Analysis in Mice.
D230
2224
3121
115
527
813
914
1911
2820
126
25B6
3216
1823
292
9
8
7
6
5
4
3
2
1
0
0.4 0.5
5
0.6
1
0.9
3
0.9
6
0.9
8
1.1
7
1.1
7
1.2
3
1.3
5
1.4
8
1.5
3
1.7
3
2.3
1
2.3
9
2.4
7
2.7
3
2.8
0
2.9
4
3.2
5
3.6
2
3.8
0
3.8
1
4.1
8
4.4
3
5.8
9
6.6
7
7.9
0
BXD Strain
Hal
op
eri
do
l - E
D 5
0 m
g/k
g
2
4
6
8
10
Inbred Strains
BA
LB/c
AK
R
D2
PL
CE
SJL
C3H
A A/H
e
B6
P CB
A
129
LP
C57L
Standard
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
20
25
30
35
40
45
50
Fli-1 (0.33)
Lap-1 (0.27)
Xmv-16 (0.40)T3d (0.40)
Ncam, D9Mit22 (0.55)D9Mit4, D9Byu1b (0.54)
Apoa-1 (0.40)
Xmv-15 (0.38)
D9Mit8 (0.04)
Drd2
11q23-q24
11q23-q2411q23-q2415q23-q2415q22-q24
(0.55)
Chromosome 9
Htr1b
d
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.
Key Publications for QTL Analysis in Mice.
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 andempirical results. Behavior Genetics 26:581-592.
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.
Key Publications for QTL Analysis in Mice.
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
Phenotype to Gene
Basic Genetics
Selective Breeding
Recombinant
InbredStrains
IntercrossMapping
Chromosome 9
cM 0 10 20 30 40 50 60 70
LO
D S
core
0
1
2
3
4
5
6
D9M
it18
8
D9M
it90
D9M
it20
6
D9M
it22
D9M
it21
D9M
it14
4
D9M
it74
D9M
it11
1
D9M
it21
2
Pe
nk2
Gria
4
El4
Grik
4S
cn2
bN
cam
Drd
2A
cat1
Acr
a5
Acr
a5
Myo
5a
Htr
1b
Acr
a3
El1
Cp
u3
Phenotype = Haloperidol-Induced Catalepsy
LO
D
0
1
2
3
4
5
6
FreeDominantRecessiveAdditive
D2M
IT80
D2M
IT464
D2M
IT521
D2M
IT241
D2M
IT458
D2M
IT94
D2M
IT420
D2M
IT102
D2M
IT491
D2M
IT493
D2M
IT412
D2M
IT282
D2M
IT229
.. . .. . . . . . . . .10 cM
IA
lcp1q
Scn1
a
Gad
1
Plcb2
Adra
2b
Sna
p2
5
Lo
rr2
Ntsr
Kcna
1-rs2N = 600
Phenotype = Ethanol-Induced Locomotor Activation
Chromosome 2
B6D2 Intercross
cM0 20 40 60 80 100
F-V
alue
0
5
10
15
20
25
30
0 - 5 Min5 - 10 Min10 - 15 Min15 - 20 Min
N = 1820 Chromosome 2
Ethanol-Induced Locomotor Activation
Comparison of IM and CIM Techniques
Relative Chromosome Length0 1
LOD
0
10
20
30
40
50
Ethanol Response 0-5 Min (CIM)
cM - Chromosome 2
0 20 40 60 80 100 120
LO
D
0
2
4
6
8
10
Free Additive
Dominant
Ethanol Response 5-10 Min (CIM)
cM - Chromosome 2
0 20 40 60 80 100 120
LO
D
0
2
4
6
8
10
Free Additive
Dominant
Ethanol Response 10-15 Min (CIM)
cM - Chromosome 2
0 20 40 60 80 100 120
LO
D
0
2
4
6
8
10
12
14
16
Free
Additive
Dominant
Ethanol Response 15-20 Min (CIM)
cM - Chromosome 2
0 20 40 60 80 100 120
LO
D
0
2
4
6
8
10
12
14
Free
Additive
Dominant
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
Evidence for epistasis – basal locomotor activity
Koyner et al. 2000
Phenotype to Gene
Basic Genetics
Selective Breeding
Recombinant
InbredStrains
IntercrossMapping
Fine
Mapping
Darvasi A (1998) Experimental strategies for the genetic dissection of complex traits in animal models. Nature Genetics 18: 19-24.
Key Publications for QTL Analysis in Mice.
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
B6D2 Intercross
cM0 20 40 60 80 100
F-V
alu
e
0
5
10
15
20
25
30
HS Population
cM
45 50 55 60 65 70 75
F-V
alu
e
-20
-10
0
10
20
30
0 - 5 Min5 - 10 Min10 - 15 Min15 - 20 Min
0 - 5 Min5 - 10 Min10 - 15 Min15 - 20 Min
Chromosome 2
N = 1820
N = 550 @ G32
Ethanol-Induced Locomotor Activation
Phenotype to Gene
Basic
Genetics
Selective Breeding
Recombinant
InbredStrains
IntercrossMapping
Fine
Mapping
Mechanism
& Circuit
Central Amygdaloid Nucleus
20
40
60
80
0 0.25 0.5 1 4
Ethanol - g/kg
2
DBA
C57
Fos
Pos
itiv
e C
ells
/ S
ecti
on
GABA(Peptide)
GABA Glu(Peptide)
DA, NE5-HT, ACh
CeL CeM
BST
Ins Cx, , PB Motor Response
GABA(Peptide)
Glu(Peptide)
1
23
5
4
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
0 20 40 60 80 100 120
-Log
P
0
1
2
3
4
5
6
C57BL/6 x DBA/2C57BL/6 x LPC57BL/6 x BALB/c
cM - Chromosome 1
0 20 40 60 80 100 120
-Log
P
0
1
2
3
4
5
6
C57BL/6 x DBA/2BALB/c x DBA/2DBA/2 x LPBALB/c x LP
C57BL/6 vs DBA/2
Chromosome 1 - cM
0 20 40 60 80 100 120
Fra
ctio
n o
f T
otal
Mar
ker
s
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
PolymorphicNot Polymorphic
Distribution of Marker Set Based On QTL Criteria
Chromosome 1 - cM
0 20 40 60 80 100 120
Fra
ctio
n of
Tot
al M
arke
rs
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Table 1: Analysis of Chromosome 1 QTL for Ethanol Response in HS Mice11
Markers (Catalog Position-cM)
Calculated Interval - cM - Log P
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
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
Gene cM F-value
glutamate receptor, ionotropic, NMDA1 (zeta 1) 12 7prostaglandin D2 synthase (21 kDa, brain) 13 12prostaglandin D2 synthase (21 kDa, brain) 13 8prostaglandin D2 synthase (21 kDa, brain) 13 7heat shock 70kD protein 5 (glucose-regulated protein, 78kD) 22 13pre B-cell leukemia transcription factor 3 22 8gelsolin 25 26tumor necrosis factor induced protein 6 30 19neurogenic differentiation 1 46 6CD59a antigen 55 50reticulocalbin 58 15secretory granule neuroendocrine protein 1, 7B2 protein 64 129maternal inhibition of differentiation 68 10beta-2 microglobulin 69 27protein tyrosine phosphata 73 6cystatin C 84 21inhibitor of DNA binding 1 84 19neuronatin 88 55protein kinase inhibitor, gamma 94 67protective protein for beta-galactosidase 96 9SRY-box containing gene 18 96 7potassium voltage-gated channel, subfamily Q, member 2 104 21myelin transcription factor 1 106 10sialyltransferase 7 7transcription termination factor 1 7c-mer proto-oncogene 9fibroblast growth factor 7 25isovaleryl coenzyme A dehydrogenase 43
Chromosome 2
S g n e1
Dif
f. S
core
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
C 5 7 B L /6 D B A /2 B A L B /c L P /J
QTL MapsEthanol Response
Chromosome 2 - cM
0 20 40 60 80 100
F va
lue
0
2
4
6
8
10
12
14
C57BL/6 x DBA/2BALB/c x DBA/2LP x DBA/2
0 20 40 60 80 100 120
F V
alue
0
2
4
6
8
10
12
14
C57Bl/6 x DBA/2C57BL/6 x BALB/cLP x BALB/cC57Bl/6 x LP
R eticu lo ca lb in
Dif
f. S
core
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
C 5 7 B L /6 D B A /2 B A L B /c L P /J
B e ta -2M ic r o g lo b u lin
Dif
f. S
core
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
C 5 7 B L /6 D B A /2 B A L B /c L P /J
Allele
Cat
alas
e A
ctiv
ity
60
70
80
90
100
110
120
130
140
B6 D2
B6RI-#1RI-#6
RI-#16D2
RI-#28
RI-#11RI-#32
D2 = C = LP Cas1
Bdnf
B6 LeuD2 MetC MetLP Leu
QTL MapsEthanol Response
Chromosome 2 - cM
0 20 40 60 80 100
F va
lue
0
2
4
6
8
10
12
14
C57BL/6 x DBA/2BALB/c x DBA/2LP x DBA/2
0 20 40 60 80 100 120
F Va
lue
0
2
4
6
8
10
12
14
C57Bl/6 x DBA/2C57BL/6 x BALB/cLP x BALB/cC57Bl/6 x LP
cM - chromosome 1
0 20 40 60 80 100 120
F v
alue
0
5
10
15
20
25
B6 vs D2LP vs D2Lp vs CC vs D2
QTL Maps - Basal Actvity
0 20 40 60 80 100 120
F-v
alue
0
5
10
15
20
25
B6 vs D2B6 vs LPB6 vs C
Gene cM F-value
Eph receptor A4 7 6
myosin Ib 25 6
acetyl-Coenzyme A dehydrogenase, long-chain 27 7
isocitrate dehydrogenase 1 (NADP+), soluble 30 6
insulin-like growth factor binding protein 5 36 23
insulin-like growth factor binding protein 2 36 8
peptidylglycine alpha-amidating monooxygenase 58 61
troponin T2, cardiac 60 11
astrotactin 1 85 7
POU domain, class 2, transcription factor 1 87 17
pre B-cell leukemia transcription factor 1 88 6
peroxisomal farnesylated protein 93 13
potassium inwardly-rectifying channel, subfamily J, member 9 94 172
ATPase, Na+/K+ transporting, alpha 2 polypeptide 94 7
LanC (bacterial lantibiotic synthetase component C)-like 104
growth arrest specific 5 61
transcription elongation factor A (SII) 1 55
kinesin heavy chain member 1A 22
expressed sequence C77892 9
Chromosome 1
cM - chromosome 1
0 20 40 60 80 100 120
F va
lue
0
5
10
15
20
25
B6 vs D2LP vs D2Lp vs CC vs D2
QTL Maps - Basal Actvity
0 20 40 60 80 100 120
F-va
lue
0
5
10
15
20
25
B6 vs D2B6 vs LPB6 vs C
Pxf
Dif
f. S
core
0
20
40
60
80
100
120
140
160
180BALB/cLP
C57BL/6DBA/2
Atp1a2D
iff.
Sco
re
0
200
400
600
800
1000
1200
1400
1600
1800BALB/cLP
C57BL/6DBA/2
GIRK3
Dif
f. S
core
0
100
200
300
400
500
600
C57BL/6DBA/2BALB/cLP
1 gggaacctag ggtactgggg gagatggtgt cagggacatg gacgccaacc cccaagggtt 61 tctgctgctg gctactcttc tctccaggct ctgggaccct gaaagcctag gaaccgactc121 tggccatcca tctctccggg aagattataa cccagagtgc ttctcagggg ggaagaattt181 gaagcaaaac cagaccccgc aggatccccg ctgcggccgc catgcgccag gagaacgccg241 ctttctctcc cgggtcggag gagccgccac gccgccgcgg tcgccagcgc tacgtggaga ………..481 gcgacctgga gcacctggag gacaccgcgt ggaccccgtg cgtcaacaac ctcaacggct541 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 …………
Polymorphisms Between the C57BL/6J and DBA/2J Strains in Kcnj9 (GIRK3)
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 + 103Gabra2 5 40cM 17.5145 0.000008 51 + 18 140 + 29 125 + 26 132 + 23Gabra6 11 23cM 13.5512 0.000047 672 + 81 848 + 89 901 + 57 922 + 71Gabrb1 5 40cM 13.5435 0.000047 965 + 85 1085 + 101 769 + 78 969 + 83Gabrb2 11* *25cM 10.0278 0.000304 34 + 14 2 + 6 44 + 17 46 + 21Gabrb3 7 29cM 1.8440 0.171715 69 + 23 83 + 22 99 + 28 86 + 17Gabrg1 5 40cM 5.8868 0.004744 40 + 8 51 + 9 64 + 14 57 + 10Gabrg2 11 19cM 3.0945 0.050183 801 + 45 881 + 72 902 + 77 888 + 54Gabrg3 7 28cM 1.3917 0.274346 71 + 25 87 + 17 80 + 11 91 + 15Gabt1 3 50cM 0.4664 0.708949 1064 + 90 1105 + 76 1110 + 65 1092 + 58Gad1 2 43cM 3.0010 0.054809 1900 + 116 1973 + 123 2055 + 123 2082 + 93
Mean + SD (N=6/strain)
Gad2 2 9cM 2.1515 0.125644 61 + 16 75 + 13 84 + 19 89 + 29
GABA Related Genes
Viaat 2 80cM 98 0.000000 1434 + 70 748 + 109 1494 + 88 1456 + 80
Gene Chr cM Region C57BL/6J DBA/2J LP/J BALB/cJ
Gabra1 11 19cM WB 526 + 44 298 + 15 505 + 59 497 + 103Striatum (DM) 272 + 106 143 + 41 195 + 55 258 + 23CeA-BSTLP 475 + 35 220 + 19 318 + 29 386 + 56
Gabra2 5 40cM WB 51 + 18 140 + 29 125 + 26 132 + 23Striatum (DM) 57 + 17 186 + 21 168 + 32 196 + 15CeA-BSTLP 108 + 10 211 + 44 217 + 24 213 + 59
Gabra3 X WB A A A AStriatum (DM) A A A ACeA-BSTLP 40 + 17 25 + 8 33 + 13 23 + 21
Gabra6 11 23cM WB 672 + 81 848 + 89 901 + 57 922 + 71Striatum (DM) 10 + 4 8 + 6 20 + 18 13 + 6CeA-BSTLP A A A A
A = absent
Mean + SD (N=3-6/strain)
Comparison of GABA Receptor Subunit Gene Expression in Whole Brain, the Dorsomedial Striatum
and the Central Extended Amygdala.
QTLs and GABA Related Gene Expression
• Chr 11 ( – Acute pentobarbital withdrawal, acute alcohol withdrawal, forced swim test, light-dark latency, tail suspension
• Chr 5 ( – 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
Genotype vs Response at D1Mit150Four-way Cross
Eth
anol
-In
du
ced
Res
pon
se -
cm
/20
min
0
1000
2000
3000
4000
5000
6000
B6:B6 C:C B6:D2 B6:LP D2:LPD2:D2 LP:LP B6:C D2:C C:LP
QTL Maps - Ethanol Response
0 20 40 60 80 100 120
-Log
P
0
1
2
3
4
5
6
C57BL/6 x DBA/2C57BL/6 x LPC57BL/6 x BALB/c
cM - Chromosome 1
0 20 40 60 80 100 120
-Log
P
0
1
2
3
4
5
6
C57BL/6 x DBA/2BALB/c x DBA/2DBA/2 x LPBALB/c x LP
Phenotype to Gene
Basic
Genetics
Selective Breeding
Recombinant
InbredStrains
IntercrossMapping
Fine
Mapping
Mechanism
& Circuit MultipleCross
Mapping
FunctionalGenomics
Complex Trait Probable Gene Reference
Intestinal tumor resistance Pla2g2a Cormier et al., 1997 Mom1 [mice] Nature Genet 17:88Type II diabetes-insulin Cd36 Aitman et al., 1999; Pravenac resistance [rats] et al., 2001, Nature
Genet 16:197 & 27:156Type I diabetes- Idd3 Lyons et al., 2000 islet degeneration [mice] Genome Res 10:446Allergen-induced airway Cd5 Karp et al., 2000 hyperresponsiveness- Nature Immun 1:221 asthma [mice]Susceptibility to lupus Ifi202 Rozzo et al., 2001 [mice] Immunity 15:435Cardiac ventricular Nppa Deschepper et al., 2001 hypertrophy [rats] Circul Res 88:223Blood pressure Cyp11b1 Cicila et al., 2001 [rats] Genomics 72:51Sweet taste preference Tas1r3 Max et al., 2001 behavior [mice] Nature Genet 28:58
JKB, Dec 2001
Complex Trait Probable Gene Method of gene ID
Intestinal tumor resistance Pla2g2a High res mapping/Transgenic Mom-1 [mice] rescueType II diabetes-insulin Cd36 Microarray analysis in congenic resistance [rats] vs background strain/
transgenic rescueType I diabetes- Idd3/Il2 Mapping to 780 kb/ islet degeneration [mice] functional testingAllergen-induced airway Cd5 Microarray analysis in congenic hyperresponsiveness-
vs background strain asthma [mice]Susceptibility to lupus Ifi202 Microarray analysis in congenic [mice] vs background strain/functionalCardiac ventricular Nppa Promotor SNP/functional hypertrophy [rats] testing/in vitro expressionBlood pressure Cyp11b1 Mapping to 220kb/SNPs/ [rats] functional testing Sweet taste preference Tas1r3 Taste cell specific expression/ behavior [mice] glycosylation site SNP/ functional
testing. JKB, Dec. 2001
Distribution of Some Murine Behavioral QTLs - LOD > 3
Chromosome
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
Num
ber
of Q
TL
s
0
5
10
15
20
25
AggressionCircadian RhythmDrug PreferenceDrug ResponseEmotionLearningMotor Activity
Total Number of QTLs = 124
Acknowledgements
Stony Brook
Jim McCaughranLaura CippKristin DemarestJay KoynerStephen Kanes Nilay PatelErik RasmussenBarbara HitzemannYifang QianPeter ThanosAdena Svingos
OHSU
Barry MalmangerShannon CoulombeStaci CooperCheryl ReedBarbara HitzemannMaureen LawlerKristin HitzemannTarra Gupta Ronnie Dhamer
Kari BuckBrooks Rademacher
Denver
Jim SikelaYan Xu
Oxford, UK
Jonathan FlintChris Talbot
