METHODS AND MATERIALS
III.3. DESCRIPTIVE OVERVIEW OF THE POLYMORPHISMS
III.4.3. Stratification by age at onset of OCD 224
III.4.3.1. Analysis of clinical variables 224
III.4.3.2. Single locus analysis of age of onset 224 III.4.3.2.1. Single locus analysis of bi-allelic loci 243 III.4.3.2.2. Single locus analysis of multi-allelic loci 243
i. DRD4 243
ii. DAT 243
III.4.3.3. Haplotype analyses: age at onset 248
III.4.3.3.1. 5-HT2Ahaplotype analysis 248
III.4.3.3.2. DRD4 haplotype analysis 248
III.4.3.3.3. COMT haplotype analysis 248
III.4.3.3.4. GRIN2B haplotype analysis 248
III.4.3.3.5. BDNF haplotype analysis 248
III.4.3.3.6. ESRα haplotype analysis 249
III.4.4. Stratification by co-morbidity 251
III.4.4.1. Major depressive disorder (MDD) 251
III.4.4.1.1. Analysis of clinical variables 251 III.4.41.2. Single locus analysis according to the presence or absence
of co-morbid MDD 252
PAGE III.4.4.1.2.1. Single locus analysis of bi-allelic polymorphisms 252 III.4.4.1.2.2. Single locus analysis of multi-allelic polymorphisms 257
i.DRD4 257
ii. DAT 257
III.4.4.2. Co-morbid tics 260
III.4.4.2.1. Clinical variables 260
III.4.4.2.2. Single locus analysis according to the presence or absence
of co-morbid tics 260
III.4.4.2.2.1. Single locus analysis of bi-allelic polymorphisms 260 III.4.4.2.2.2. Single locus analysis of multi-allelic polymorphisms 262
i.DRD4 262
ii. DAT 262
III.4.5. Stratification by primary symptom dimension 268
III.4.5.1. Hoarding symptoms 268
III.4.5.1.1. Analysis of clinical variables 268 III.4.5.1.2. Single locus analysis of hoarding symtomatology 270 III.4.5.1.2.1. Single locus genetic investigation of bi-allelic polymorphisms 270 III.4.5.1.2.2. Single locus analysis of multi-allelic loci 271
i. DRD4 271
ii. DAT 271
III.4.5.2. Symmetry and ordering symptoms 277
III.4.5.2.1. Analysis of clinical variables 277 III.4.5.2.2. Single locus analysis of symmetry/ordering symptomatology 277 III.4.5.2.2.1. Single locus genetic investigation of bi-allelic
polymorphisms 277
III.4.5.2.2.2. Single locus analysis of multi-allelic polymorphisms 278
i. DRD4 278
ii. DAT 278
III.4.5.3. Sexual and religious obsessions and / or compulsions 285 III.4.5.3.1. Analysis of clinical variables 285 III.4.5.3.2. Single locus analysis of sexual/religious symptomatology 287 III.4.5.3.2.1. Single locus genetic investigation of bi-allelic polymorphisms 287
PAGE III.4.5.3.2.2. Single locus analysis of multi-allelic polymorphisms 291
i. DRD4 291
ii. DAT 291
III.4.5.4. Contamination Symptoms 294
III.4.5.4.1. Analysis of clinical variables 294 III.4.5.4.2. Single locus analysis of contamination symptomatology 294 III.4.5.4.2.1. Single locus analysis of bi-allelic polymorphisms 294 III.4.5.4.2.2. Single locus analysis of multi-allelic polymorphisms 299
i. DRD4 299
ii. DAT 299
III.4.5.5. Aggressive Symptoms 304
III.4.5.5.1. Analysis of clinical variables 304 III.4.5.5.2. Single locus analysis of aggressive symptomatology 304 III.4.5.5.2.1. Single locus genetic investigation of bi-allelic polymorphisms 304 III.4.5.5.2.2. Single locus analysis of multi-allelic polymorphisms 309
i. DRD4 309
ii. DAT 309
III.5. META-ANALYSES 312
III.5.1. 5-HT2A -1438 A/G (rs6311) 312
III.5.2. 5-HT2A T102C (rs6313) 312
III.5.3. 5-HT2C cys23ser(rs6318) 315
III.5.4 DAT 40bp VNTR 315
III.5.5. DRD2 Taq1A (rs1800497) 319
III.5.6. DRD3 ser9gly 319
III.5.7. COMT val158met (rs4680) meta-analysis 322
III.5.8. DRD4 48bp VNTR 326
CHAPTER III: RESULTS
III.1. GENOTYPING RESULTS
All polymorphisms were genotyped as described in Chapter II. The following figures (Figure III.1-III.37) depict representative gels for each polymorphism.
1 2 3 4
Figure III.1. ASREA of the 5-HT2A -1438A/G (rs6311) polymorphism. Representative 2.5%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with MspI. Lane 1: -1438A/-1438A; Lane 2: -1438G/-1438G; Lane 3: -1438A/-1438G; Lane 4:
Molecular weight marker X.
1 2 3 4 5
Figure III.2. ASREA of the 5-HT2A T102C (rs6313) polymorphism. Representative 2.5%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with HpaII. Lanes 1 and 3: C102C; Lane 2: T102C; Lane 4: T102T; Lane 5: Molecular weight marker X.
468bp 244bp
224bp
372bp
216bp 156bp
1 2 3 4
Figure III.3. ASREA of the 5-HT1Dβ G861C (rs6296) polymorphism. Representative 2%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with HincII. Lane I: C861C; Lane 2: G861G; Lane 3:G861C; Lane 4: Molecular weight marker X.
1 2 3 4 5
Figure III.4. ASREA of the 5-HT6 T267C (rs1805054) polymorphism. Representative 2.5%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with RsaI.
Lane I: T267T; Lanes 2 and 3: T267C; Lane 4: C267C; Lane 5: Low molecular weight DNA ladder.
662bp
482bp
180bp
578bp 449bp
129bp
1 2 3 4 5 6
Figure III.5. ASREA of the 5-HT2C cys23ser (rs6318) polymorphism. Representative 3%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with NlaIII. Lane I: undigested PCR product (control); Lane 2: ser23ser; Lanes 3 and 4:
cys23cys; Lane 5: cys23ser; Lane 6: Molecular weight marker X.
1 2 3 4
Figure III.6. ASREA of the DRD4 -521C/T (rs1800955) polymorphism. Representative 2.5% agarose gel showing the fragment sizes generated by PCR and restriction digestion with FspI. Lane I: -521C/C; Lane 2: -521C/T; Lane 3: -521T/T; Lane 4: Molecular weight marker X.
184bp 150bp 130bp
380bp
228bp 180bp
1 2 3 4 5 6 7
Figure III.7. PCR amplification of the DRD4 48bp VNTR polymorphism. Representative 3% agarose gel showing the fragment sizes generated by PCR. Lane 1: A2/A2; Lane 2:
A4/A4; Lanes 3 and 6: A4/A3; Lane 4: A4/A7; Lane 5: A6/A7; Lane 7: Molecular weight marker XIV. (A2=379bp, A3=427bp, A4=475bp, A6=571bp, A7=619bp).
1 2 3 4
Figure III.8. ASREA of the DRD2 Taq1A (rs1800497) polymorphism. Representative 2.5%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with Taq1A I. Lane 1: CC; Lane 2: CT; Lane 3: TT; Lane 4: Marker XIV
500bp
130bp 180bp 310bp
1 2 3 4
Figure III.9. ASREA of the COMT promoter (rs2097603) polymorphism. Representative 2.5% agarose gel showing the fragment sizes generated by PCR and restriction digestion with HindIII. Lane 1: AG; Lane 2: GG; Lane 3: AA; Lane 4: Molecular weight marker X.
1 2 3 4
Figure III.10. ASREA of the COMT val158met (rs4680) polymorphism. Representative 12% polyacrylamide gel showing the fragment sizes generated by PCR and restriction digestion with NlaIII. Lane 1: AA (met158met); Lane 2: GG (val158val); Lane 3: AG (val158met); Lane 4: Molecular weight marker λPst.
306bp 271bp
75bp
82bp 68bp
1 2 3 4
Figure III.11. ASREA of the COMT exon 6 (rs362204) polymorphism. Representative 2.5% agarose gel showing the fragment sizes generated by PCR and restriction digestion with BglI. Lane 1: ID (C+/C-); Lane 2: DD (C-/C-); Lane 3: II (C+/C+); Lane 4: Molecular weight marker λPst.
1 2 3
Figure III.12. ASREA of the DRD3 ser9gly (rs6280) polymorphism. Representative 3%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with MscI. Lane 1: AA (ser9ser); Lane 2: AG (gly9ser) Lane 3: Molecular weight marker XIV.
The visible constitutive fragments (at 47bp and 146bp) are marked with an asterisk.
GG (gly9gly) is not indicated on the gel.
278bp 196bp 82bp
304bp 206bp 111bp**
98bp 47bp**
1 2 3 4 5
Figure III.13. ASREA of the DRD1 A-48G polymorphism. Representative 2.5% agarose gel showing the fragment sizes generated by PCR and restriction digestion with DdeI. Lane 1: -48G/-48G; Lane 2: -48A/-48G; Lane 3: -48A/-48A; Lane 4: undigested PCR product; Lane 5: Molecular weight marker X.
The constitutive fragment (at 146bp) is marked with an asterisk.
1 2 3
Figure III.14. ASREA of the BDNF val66met (rs6265) polymorphism. Representative 3%
agarose gel showing the fragment sizes generated by PCR and restriction digestion with NlaIII. Lane 1: GG (val66val); Lane 2: GA (val66met); Lane 3: Molecular weight marker XIV.
The constitutive fragment (at 57bp) is marked with an asterisk.
AA (met66met) is not indicated on this gel.
259bp 217bp 146bp*
42bp 405bp
217bp 140bp 77bp 57bp*