Transcript Integration of QTL, Gene Expression and Sequence Analyses

Integration of QTL, Gene Expression and
Sequence Analyses: A Pathway to the
Detection of Quantitative Trait Nucleotides.
Robert Hitzemann, Ph.D.
Department of Behavioral Neuroscience
Oregon Health and Science University
25
Number of QTLs
20
15
Distribution of Mouse
Behavioral QTLs - LOD > 3
Total Number of
QTLs = 125
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
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
LPxB6 F2
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)
Multiple Cross Mapping
C57BL6/J (B6)
B6xD2
DBA/2J (D2) BALB/cJ (C) LP/J (LP)
B6xC
B6xLP CxLP D2xLP D2xC
Four Inbred Strains  Six F2 Intercrosses
Most suitable for known QTLs
Multiple Cross Mapping (MCM)
• Built from the observation that for open-field/basal
activity, three different diallele crosses (B6xC, B6xA and
B6xD2) appeared to generate similar QTLs, most notably
on distal chromosome1 (Hitzemann et al. 2000). We
interpreted these data to indicate that the C, A and D2
strains have common allele(s) in the region of interest.
• The more general interpretation was that polymorphic and
non-polymorphic alleles are not randomly distributed and
thus, provide a source of information. MCM could be used
to “mine” this information (haplotype structure) to reduce
the QTL interval (Hitzemann et al. 2002, 2003).
• MCM provides a mechanism to interrogate the data
obtained from gene and protein array analysis.
• Leverages the most successful aspect of QTL analysis –
namely QTL detection.
How Does MCM Work?
A
B
C
D
QTL is detected in AxB, AxC and AxD crosses but not in
BxC, BxD and CxD intercrosses.
1600
Figure 1C
1400
1200
1000
800
Genotype
C:C
B6:C
B6:B6
LP:LP
B6:C
B6:B6
D2:D2
B6:D2
B6:B6
7
Figure 1B
B6xD2
6
B6xC
5
- Log P
Step 1: QTL analyses for
basal locomotor activity in
B6xC, B6xLP, CxLP, D2xC
and D2xLP intercrosses.
Distance - cm/2.5 min
Known: QTL for basal
locomotor activity found
in B6xD2 intercross
(Koyner et al. 2000).
4
B6xLP
3
2
Result: QTL detected in all
B6 intercrosses. B6 allele
increases activity.
1
0
7
Figure 1A
Figure 1A
6
- Log P
5
4
CxLP
3
2
1
D2xC
0
140
D2xLP
150
160
170
Chr 1 - Mb
180
190
200
All Markers - N = 106
5
3
Step 2: Sequentially sort the
the Mit microsatlellite markers
over the region of interest on
the basis of the data obtained
in step 1.
1
B6 vs D2 - N = 72
5
3
1
B6 vs (D2 & C) - N = 50
5
Result: The markers segregate
into three domains. The domain
centered at 175 Mb appears to
be the most congruent with the
QTL analyses.
Markers/Mb
3
1
B6 vs (D2, C & LP) - N = 46
5
3
1
5
B6 vs (D2, C & LP); C = LP - N = 18
3
1
4
3
B6 vs (D2, C & LP); C = LP; D2 =LP - N = 12
2
1
0
160
170
180
Mb - Chromosome 1
190
250
200
50
B6 vs A
250
SNPs/10Kb
Results: SNP databases for the
all of the strains of interest were
not available. Data were available
for the B6, D2 and A strains. It
was known that a B6xA cross
also produced a QTL in the
region of interest (Gershenfeld
et al. 1997). The results obtained
were very similar to those found
with the SSLPs – the region
showing the highest density of
polymorphisms was found
between 170 and 175 Mb.
B6 vs D2
0
200
50
0
250
B6 vs (D2 & A)
SNPs/10Kb
Step 2a: Validate the segregation
of the microsatellite markers
using SNPs.
SNPs/10Kb
300
200
50
0
16000
16500
17000
17500
18000
Basepairs/10
4
18500
19000
7
6
QTL Map for Basal Activity in
HS/Npt Mice (G35)
5
-Log P
Step 3: Validate the location of
the QTL as determined by the
segregation of the SSLP and
SNP markers. Strategy – map the
QTL using heterogenous stock (HS)
and congenic (B6.D2Mtv) mice.
4
3
2
1
0
cM 75
Mb
Mb
80
85
160
150
90
95
170
1500
1000
500
0
B6:B6
B6:Mtv
Mtv:Mtv
100
180
2000
Distance(cm)/2.5 min
Results: Mapping in HS animals
confirms that the QTL is located
between 170 and 175 Mb. The black
bar indicates the extent of the
introgressed interval which captured
the QTL.
D2:D2
Step 4: Examine gene
expression in the region of
interest. Six genes within
the interval were differentially expressed at p< 10-6
or better – this rate of
differential expression is
approximately 5 times the
genome wide rate.
Genes showing significant
differential expression Aldh9a1,Mgst3,Rgs5, Sdhc,
Kcnj9 and Dfy.
Gene
Table 1: Whole Brain Gene Expression -Chromosome 1 (168 to 178 Mb).
Mean Signal + SD
Mb
C57Bl/6J
DBA/2J
LP/J
BALB/cJ
Uck
168.40
1190006A08RIK
168.50
Aldh9a1
168.60
Mgst3
168.70
Pbx
169.40
Rgs5
170.96
Rgs4
171.02
Eef1bl
171.47
Dusp12
172.16
Sdhc
172.41
Nr1i3
172.50
Ndufs2
172.51
B4galt3
172.55
Usp23
172.56
1110021H02Rik172.57
Dedd
172.62
Nit1
172.62
Usf1
172.70
Jcam1
172.74
Copa
173.41
Pxf
173.42
AA408877 173.49
Pea15
173.49
Atp1a2
173.56
Igsf8
173.61
Kcnj9
173.61
Pigm
173.67
Tagln2
173.80
Dfy
174.63
Prdc
176.15
Rgs7
176.37
Fh1
176.92
Rbm8
177.29
93
218
125
1058
833
121
145
1307
141
1152
138
1378
334
151
293
57
151
97
93
921
260
222
803
1362
1146
162
169
422
209
64
448
546
166
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
6
9
5
24
22
12
17
44
10
57
4
226
14
14
33
6
29
17
9
120
23
5
99
93
37
12
14
87
14
4
44
18
31
83
260
32
1588
949
131
160
1344
152
1459
123
1573
304
153
274
73
153
83
125
716
266
204
713
1424
1179
801
149
393
149
81
344
566
204
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
4
16
3
46
46
9
11
80
23
39
5
79
36
14
21
3
30
9
11
53
8
13
60
198
87
39
15
33
8
8
54
35
35
89
246
125
1037
831
42
157
1298
141
1456
131
1217
306
176
292
74
141
132
96
744
253
209
675
1290
1073
880
165
406
160
67
364
492
199
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
6
15
6
36
51
6
16
146
16
46
8
49
22
15
37
3
12
10
14
143
21
12
51
43
38
45
16
70
9
9
28
64
24
96
251
113
1023
796
167
180
1449
127
1062
139
1077
279
195
265
69
134
121
109
777
243
192
662
1188
1000
832
155
421
175
69
338
546
198
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
6
20
13
39
53
11
24
57
11
50
9
51
20
48
22
11
15
9
1
125
24
4
63
35
55
71
27
81
14
5
27
38
28
F value
1.1
1.3
32.7
55.3
2.8
101.1
2.5
2.1
1.5
65.6
4.3
10.5
3.1
2.1
0.8
5.1
0.5
13.3
7.4
2.2
0.9
6.4
2.9
2.9
6.9
190.7
0.9
0.1
18.5
4.4
5.8
2.0
1.2
p < 0.0000001
p < 0.00001
p < 0.0001
p < 0.001
p < 0.01
p > 0.01
200
100
Kcnj9: potassium inwardly-rectifying channel,
subfamily J, member 9 (GIRK3)
Myoc: myocillin
Srp9: signal recognition particle 9 kDa
F Score
Tcea 1: transcription elongation factor A (SII) 1
Pam: peptidylglycine alpha
amidating monooxygenase
Mgst3: microsomal glutathione S-transferase 3
Rgs5: regulation of
G-protein signaling 5
Lancl1: Lan C (bacterial lantibiotic
synthetase component C)-like
50
AU024699
D1Ert202, DNA segment, Chr 1,
ERATO Doi 202, expressed
Cbx3, chromobox homolog 3
(Drosophila HP1 gamma)
Hspe1: heatshock 10 kDa
protein 1 (chaperonin 10)
A
C
D
B
Sdhc: succinate dehydrgenase
complex, subunit C, integral
Pou2f1: POU domain,
membrane protein, 15 kDa
class 2, transcription factor 1
Cop1: constitutive photomorphic
G
protein 1 (Arabidopsis)
I
F
E
H
J
0
0
10
20
30
40
50
60
70
80
90
cM Location on Chromosome 1
100
110 Syntenic
Chromosome 1
B6-D2 B6-LP B6-C D2-LP D2-C C-LP
20cM
40cM
60cM
80cM
100cM
=> 1.4 fold
= < 1.4 & > 0.72 fold
= < 0.72 fold
Gene
Table 2. Dorsomedial Striatum Gene Expression - Chromsome 1 (168-178 Mb)
Mean Signal + SD
Mb
C57Bl/6J
DBA/2J
LP/J
BALB/cJ
Uck
168.40
1190006A08RIK
168.50
Aldh9a1
168.60
Mgst3
168.70
Pbx
169.40
Rgs5
170.96
Rgs4
171.02
Eef1bl
171.47
Dusp12
172.16
Sdhc
172.41
Nr1i3
172.50
Ndufs2
172.51
B4galt3
172.55
Usp23
172.56
1110021H02Rik172.57
Dedd
172.62
Nit1
172.62
Usf1
172.70
Jcam1
172.74
Copa
173.41
Pxf
173.42
AA408877
173.49
Pea15
173.49
Atp1a2
173.56
Igsf8
173.61
Kcnj9
173.61
Pigm
173.67
Tagln2
173.80
Dfy
174.63
Prdc
176.15
Rgs7
176.37
Fh1
176.92
Rbm8
177.29
89
237
473
840
540
131
577
836
164
1444
143
1415
284
161
315
75
111
102
104
795
234
203
980
920
1020
93
145
355
73
88
336
607
168
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3
23
64
140
137
7
170
135
15
181
8
181
24
15
31
18
6
47
12
124
71
25
63
104
94
21
20
31
33
26
41
50
20
83
282
271
907
859
166
613
1024
150
1879
159
1699
266
158
289
94
117
102
123
573
232
168
1020
957
1027
397
150
426
101
105
341
596
180
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
10
46
23
90
42
49
218
55
18
128
5
246
31
31
32
30
12
7
25
80
44
12
95
122
174
59
21
69
16
11
49
29
11
98
245
279
893
651
61
474
932
151
1918
155
853
231
155
252
77
119
134
111
604
163
169
1105
879
901
318
130
453
74
93
325
542
165
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
16
46
60
240
63
8
51
107
13
116
24
73
30
13
18
10
6
15
12
39
22
2
69
68
106
45
25
10
23
30
19
28
28
74
251
256
750
763
150
507
1001
183
1389
153
1257
230
163
268
67
106
139
81
604
187
167
836
843
986
276
122
379
107
119
266
612
192
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
16
14
56
44
44
59
293
70
29
34
24
270
20
29
15
5
4
18
4
148
35
19
109
114
41
50
14
24
18
11
98
94
20
Table 3: Central Extended Amygdala Gene Expression - Chromosome 1 (168-178 Mb)
Mean Signal + SD
Gene
Mb
C57Bl/6J
DBA/2J
LP/J
BALB/cJ
Uck
168.40
1190006A08RIK
168.50
Aldh9a1
168.60
Mgst3
168.70
Pbx
169.40
Rgs5
170.96
Rgs4
171.02
Eef1bl
171.47
Dusp12
172.16
Sdhc
172.41
Nr1i3
172.50
Ndufs2
172.51
B4galt3
172.55
Usp23
172.56
1110021H02Rik172.57
Dedd
172.62
Nit1
172.62
Usf1
172.70
Jcam1
172.74
Copa
173.41
Pxf
173.42
AA408877
173.49
Pea15
173.49
Atp1a2
173.56
Igsf8
173.61
Kcnj9
173.61
Pigm
173.67
Tagln2
173.80
Dfy
174.63
Prdc
176.15
Rgs7
176.37
Fh1
176.92
Rbm8
177.29
103
283
163
960
1031
88
296
156
114
1235
114
1508
296
159
356
57
163
79
86
1076
234
216
992
741
870
117
152
318
130
113
535
739
184
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
27
6
4
65
107
48
89
16
12
131
17
46
1
26
53
4
10
2
5
35
31
48
63
59
87
4
13
39
8
10
22
125
3
81
295
87
1429
919
74
180
143
148
1379
118
1691
253
150
300
77
100
66
99
930
190
211
932
823
1103
426
151
360
92
116
347
691
203
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3
38
17
137
44
23
58
20
41
130
19
145
46
28
36
8
8
26
20
84
12
19
118
73
67
48
18
55
5
25
31
9
17
101
245
125
905
923
37
192
157
148
1286
119
1179
237
158
293
58
123
125
90
904
224
170
1013
677
889
327
145
304
76
121
342
659
231
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
10
19
26
87
15
85
34
16
61
13
116
71
28
33
16
11
27
1
35
15
14
116
101
9
105
43
34
32
27
55
55
36
88
347
116
1185
866
95
265
172
201
1198
129
1167
225
161
281
61
144
118
90
915
178
176
856
680
828
412
151
379
123
140
417
612
169
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
20
127
37
36
68
10
61
41
10
68
14
129
4
28
18
16
20
8
7
84
37
17
58
28
58
52
10
14
10
8
56
73
5
Table 4: QTL Analysis for Kcnj9 Expression.
Step 5: Within the region of
interest only Kcnj9 shows the
appropriate (MCM) pattern of
differential expression. If Kcnj9
is the QTG, then is should
exhibit apparent cis-regulation.
Results: Using the WebQTL
mapping tool and the BXD RI
transcriptome database, Kcnj9
does exhibit apparent cisregulation. In addition,
potential trans-regulatory
QTLs were detected on Chrs
3, 4, 18 and 19.
Marker
D1Mit200
D1Mit425
D1Mit105
D1Mit106
D1Mit145
P1Ehs1
Mpmv25
D1Mit113
D1Mit150
D1Mit426
Mb
151.2
157.7
161.6
163.5
168.5
N/A
N/A
173.1
175.9
181.8
LRS
18.63*
13.99
18.63*
12.88
17.31
18.63*
25.92*
62.99**
33.88**
24.46*
Coefficient
0.501
0.453
0.501
0.432
0.484
0.501
0.567
0.698
0.623
0.549
D3Mit12
D3Mit347
100.6
N/A
10.46
10.8
0.402
0.404
D4Mit135
19.4
12.57
0.428
D18Mit19
Iapls3-5
D18Mit31
D18Mit83
4.9
N/A
11.1
20.9
10.69
17.99
14.44
20.71*
0.401
0.493
0.458
0.531
D19Mit68
7.2
11.74
0.437
r -point biserial correlation
0.8
Mapping QTLs forKcnj9Expression BXD RI Series
p < 0.001
0.7
Chr 11
Chr 1
Chr 18
Chr 15
0.6
p < 0.01
0.5
0.4
0.3
cM
0
200
400
600
800
1000 1200 1400 1600
Table 5: Nucleotide Changes Within the Coding Region of Kcnj9
Nucleotide Change
SNP Sequence Context
A270G
CTG GA(A/C) CAC CTG
G378C
CAC CG(G/C) GTC ATC
G594A
TCC TC(G/A) CAC ATC
C618A
ATC CG(C/A) GCC CTC
G918C
ACA TC(G/C) GTG CTC
C1035T
CGC CT(C/T) GAT GCC
Step 6: Examine the coding and non-coding regions of the candidate QTG
For polymorphisms that match the MCM algorithm.
Results: Within the coding region there are six SNPs between the B6 and D2
strains – no effect on AA composition but they affect transcriptional efficiency.
Step 6: Use the MCM algorithm to detect relevant polymorphisms in
transcription binding sites that are likely to affect Kcnj9 expression.
STRAIN
DBA
C57
BALB/C
LP
SEQUENCE
C
C
C
C
A
A
A
A
G
G
G
G
T
T
T
T
T
C
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
A
A
A
A
C
C
C
C
A
A
A
A
Predicted Transcription Factor Binding Site Disruptions:
Ikaros 1
MZF1
C/EBPbeta
22 24 24 24 22
T T C C C
18 20 19 18 20
T C C C C
21 20 0 21 5
T T C C C
Although there are 3 predicted TRANSFAC sites disrupted by the
SNP, the Ikaros 1 site and Mzf1 site seem to be most disrupted by
the bp difference.
C
C
C
C
# of seq.
24
20
21
Step 7: Independently confirm differences in gene
expression and/or sequence e.g. RT-PCR. Confirm
QTN or QTNs within promoter region affect
expression.
Step 8: Create BAC-transgenic mice to confirm
the QTG.
Steps 9 and beyond: Especially when step 8 is not
possible, use available methods to confirm the QTG.
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
Tennessee
Kari Buck
Nicole Walter
Brooks Rademacher
Jonathan Flint
Chris Talbot
John Belknap
Roswell Park
Rob Williams
Oxford, UK
Ken Manly
9860.02799
9536.09278
9212.15757
8888.22236
8564.28714
8240.35193
7916.41672
7592.48150
7268.54629
6944.61108
6620.67586
6296.74065
5972.80544
5648.87023
5324.93501
<=
60
50
40
30
20
10
0