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The DRD4 was examined for polymorphisms in association with schizophrenia in the EA population using the non-isotopic SSCP method. Seven DNA fragments o f the DRD4 were screened for mutations and these have been listed in table 3.3 (see figure 3.1). The results of the SSCP analysis are summarised in table 3.3.

The majority o f patient DNA samples were examined for polymorphisms in the DRD4. DNA fragments that did not exhibit different SSCP banding patterns between patients and controls, hence the absence o f nucleotide variation between patients and controls, were only examined in a small number o f control DNAs. It must also be noted that not all seven DNA fragments could not be amplified from all the patient and control DN A samples, probably due to degradation o f the genomic DNAs.

The D2-like dopamine receptors Table 3.3: Results of SSCP analysis of the DRD4.

PC R amplified Fragments N um ber of DNA samples screened Results of SSCP analysis D 4E xl+/D 4E xlB - 66 Subjects 68 Controls

Numerous bands - 10 out of 68 controls and 8 out of 66 cases exhibited a different banding pattern compared to the rest of the DNAs (see figure 3.2a)

D4Ex 1C+/ D4Ex 1 - 70 Subjects 55 Controls

2 Distinct bands were observed - no polymorphism detected (see figure 3.2b)

D4Ex2+/ D4Ex2- 60 Subjects 20 Controls

A number of distinct bands were observed in both control and patient subjects (see figure 3.2c) - no polymorphism associated with disease was detected.

D4In2+/ D4In2- 50 Subjects 20 controls

2 Bands were observed - no polymorphism detected

D4Ex3+/ D4Ex3- 60 Subjects 20 controls

The SSCP run was smeary - but a number of distinct bands were visible - no polymorphism associated with disease was detected

D4Ex4-5+/ D4Ex4-5- 70 Subjects 20 controls

Two distinct bands were observed - no polymorphism detected

D4Ex5+/ D4Ex5- 60 Subjects 20 controls

The SSCP run was smeary but two bands were visible - no polymorphisms detected

O f the seven DNA fragments only one, the D 4Exl+/D 4ExlB-, exhibited different SSCP banding patterns suggestive o f a polymorphism (see figure 3.2a). The nature o f the polymorphism was established by sequencing a minimum o f 5 individuals for each o f the two different banding patterns generated. The y-^^P-dATP end labelled primer sequencing protocol was used, the sequence data has not been presented. The DRD4 exonl sub-fragment SSCP band shift was caused by a 12bp deletion. This polymorphism has been previously reported and shown not to be associated with schizophrenia (see section 1.12.2.2). HD analysis was performed on this PCR amplified fi-agment from 66 patient and 68 control subjects. The heteroduplex analysis o f this fi*agment also showed the same frequency o f heterozygotes as the fi*equency o f band shifts observed in the SSCP analysis in the case and control subjects, thereby confirming the results o f the SSCP analysis (see figure 3.3a). The formation o f heteroduplexes suggests these individuals are heterozygous for the mutation.

The frequency o f this mutation was determined to be 12.1% in case and 14.7% in control subjects. The basic 2x2 Chi squared test (%^) was applied to assess the level o f significance o f the differences in the frequency o f the genotype o f the exonl polymorphism between case and control subjects. A value o f 0.147 and a significance

1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 1 4 1 5 16 17 (a)

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Figure 3.2: SSCP results of the human DRD4. (a) SSCP banding patterns observed for fragment l(D 4Exl+/ D4ExlB-). Lanes 1, 2, 3, 4, 5, 7, 8, 10, 12, 13, 15, and 16 - individuals homozygous for allele 1 (2 fold 12bp repeat). Lanes 6, 9, 11, 14 and 17 - individuals heterozygous for the 12bp repeat, with 1 fold and 2 fold repeats. NDC refers to non-denatured controls, (b) SSCP banding pattern for fragment 2 (D4ExlC-i-/D4Exl-). All individuals examined showed the same banding pattern, polymorphisms were not identified in this fragment, (c) SSCP banding pattern for fragment 3 (D4Ex2+/ D4Ex2-). All individuals examined showed the same banding pattern, polymorphism in assocition with disease was not identified in this fragment.

1 2 3 4 5 6 7 8 9 1011 1213 14 15 1617 18 Single stranded DNA fragments Double stranded DNA duplexes

Figure 3.3a: HD result of fragment 1 (D4ExI+/ D4ExlB-) of the human DRD4.

Lanes 1, 2, 4, 6 to 13 and 15 to 18; Homoduplexes of the 2 fold 12bp repeat allele (allele 1)

Lanes 3, 5 and 14; Heteroduplexes representing the 2 fold 12bp repeat and the deletion mutant single 12bp repeat allele (allele 2).

The single stranded bands in the polyacrylamide gel are those that failed to form duplexes during the cooling stage, after PCR amplification of fragment 1 (see section

2 10.2).

Double stranded DNA duplexes

Figure 3.3b: HD result of fragment 2 (D 4ExlC+/ D 4E xl-) of the human DRD4. The lanes represent homoduplexes, showing the absence of a polymorphism in this fragment.

The D2-like dopamine receptors value o f 0.7 was obtained, suggesting a non signifieant difference, and therefore no association with disease.

The exonl sub-fragment, D 4ExlC +/D 4Exl-, which did not show DNA band shifts in the SSCP analysis was also screened by HD method. This was perform ed essentially to confirm the absence o f a 13bp deletion that has been identified in this fragment by other studies in other populations (see section 1.12.2.2). However this deletion has been reported not to be associated with schizophrenia in these studies. Heteroduplexes were not observed in the EA population sample, consistent with the results o f the SSCP analysis (see figure 3.3b).