SSCP ANALYSIS

PCR products representing exons 1, 2 and 3 were analysed by SSCP electrophoresis. No novel m utations w ere identified in the SA patients, although, it was possible to genotype all samples for the previously identified com m on variants in exon 2 (S erl21S er; section 3.4.1) and exon 3

(G lyl77A sp; section 3.4.2). Genotypes were checked by restriction enzym e digestion with P vu ll (exon 2) and Bam H l (exon 3). In addition, H a elll

digestion was used to type the TIVSyC polym orphism (section 3.1).

Conditions for SSCP analysis of exons 4, 5, 6, 7 and 8 w ere explored in the same way as described for exons 1, 2 and 3 (section 3.3 and 3.4); the conditions finally chosen for maximal resolution are shown in Table 4.1 (the 5'U TR was not analysed by SSCP). For exon 6, w here the am plified product is 223 bp, the product was digested with P vu ll to give two fragm ents o f 119 bp and 104 bp, and to allow a thorough SSCP analysis of sm aller DNA fragm ents (Fig 4.1 B).

No m utations are present in exons 4, 5, and 6 in either the controls or SA patients (Fig 4.1 and 4.2). However, exon 7 showed a com m on

polym orphic variation (Fig 4.3) which could be explained by the presence of two alternative alleles at a single site. In Fig 4.3 samples 27, 32, 33, 34 and 35 are hom ozygous for the com m on allele, while individuals 2 8 -3 1 are

E x o n % T % C V olts T im e (hrs) # 1 10 1.5 300 6.5 2 10 1.5 300 6.0 3 10 1.5 300 2.5 4 10 2.5 300 2.5 5 10 2.5 300 5.0 6 10 2.5 300 5.0 7 12 2.5 150 16.0 8 10 2.5 300 19.5 8(F/Rb) 10 2 310 4.5 8(Fb/R)* 10 1.5 150 20.0

T a b le 4.1 Conditions for SSCP analysis of the hum an T gene, t indicates that only the coding sequence of exon 1 was analysed and * indicates the use of 5% glycerol in the gel. All gels are run at room tem perature exept for exon 1 ($) which is run at 4°C.

B

2 3 4 5 6 7 8

1

— 119 bp—I

— 104 bp J ds D N A

Figure 4.1 SSCP analysis of T exon 6 in sacral agenesis patients and their parents (samples 2-8). A. Analysis of indigested samples. Electrophoresis was performed on a 10%T, 2.5%C polyacrylamide gel in 0.5x TBE at room temperature for 5 hours at 300 Volts. B. Analysis of PvuW digested PCR products.

Electrophoresis was carried out on a 10%T, 2%C polyacrylamide gel in 0.5x TBE at room temperature for 2 hours at 300 Volts. The double stranded DNA products of the PvuW digestion are indicated.

B

Figure 4.2 SSCP analysis of T exon 4 (A) and exon 5 (B) in sacral agenesis patients and their parents (samples 1-6). Electrophoresis was performed on a 10%T, 2.5%C polyacrylamide gel in 0.5x TBE at room temperature, 300 Volts for 2.5 hours (exon 4) and 5 hours (exon 5).

27 28 29 30 31 32 33 34 35

Figure 4.3 SSCP analysis of T exon 7 in sacral agenesis patients and their parents (samples 27-35). Electrophoresis was perfonned on a

12%T, 2.5%C polyacrylamide gel in 0.5x TBE at room temperature, 150 Volts for 16 hours. The genotype assessed by sequence analysis is shown above lanes.

heterozygous for the com m on and variant alleles. The nucleotide change underlying this variant was identified by direct sequencing (see next section).

In the case of exon 8, where a 338 bp PCR fragm ent is generated, the SSCP analysis was complex. Close exam ination o f the banding pattern suggests that two, if not three common variants occur, which are difficult to resolve. Fig 4.4 A shows a group of 10 individuals selected to show the variety o f banding patterns seen. Some relationships were easier to interpret. For example, if the banding patterns in the top half of the gel in Fig 4.4 A are exam ined, samples 3 and 4 look hom ozygous for one type of allele, 8 and 9 hom ozygous for a second allele and 1, 2 and 5 heterozygous com binations of the two alleles. Similarly, on the bottom half of the gel, samples 2 , 5 , 8 and 9 are hom ozygous for one type of allele, sample 4 for another and 1 and 3 are the corresponding heterozygotes.

Before attempting any further typing, it was decided to design new internal prim ers (TX8Fb and TX8Rb; see Table 2.1 and A ppendix I). These could be used in com bination with primers TX 8F and TX 8R to generate two overlapping PCR products, which span the entire exon 8. The TX 8F/TX 8Rb product is 159 bp and contains the 5' end of exon 8 (referred as the exon 8-5' product) and the TX 8Fb/TX 8R product is 2 5 6 bp and contains the 3' end o f exon 8 (referred as the exon 8-3' product). SSCP analysis of the exon 8-5' product showed a banding pattern that could be explained by the presence of three different alleles at a single site. In Fig 4.4 B, which shows the analysis of 10 selected control individuals, sample 2 is hom ozygous for the type I allele, sample 9 is hom ozygous for the type 2 allele, samples 3, 4, 7, 8 and 10 appear to be 2.1 heterozygotes and samples I, 5 and 6 are apparently

heterozygotes com bining the type 2 allele w ith a third allele, type 3. No individuals hom ozygous for the type 3 allele w ere seen.

Fig 4.4 C shows the analysis of the 3' product o f exon 8. The banding pattern suggests the presence of two alleles at a single site. For exam ple, samples 1 and 2 are homozygous for the type I allele, sample 7 is

1 2 3 4 5 6 7 8 9 10

W W ^

m m

Figure 4.4 SSCP analysis of T exon 8 in selected British controls. Samples numbers do not represent the same individual in each gel. A.

Samples amplified using the TX8F/TX8R primers (product 338 bp) were electrophoresed on a 10%T, 2.5%C polyacrylamide gel in 0.5x TBE at room temperature for 19.5 hours at 300 Volts. B. Analysis of the TX8F/ TX8Rb 5 ’product (159 bp). Samples were electrophoresed on a 10%T, 2%C polyacrylamide gel in 0.5x TBE at room temperature, 310 Volts for 4.5 hours. C. Analysis of the TX8Fb/TX8R 3 ’ product (256 bp).

Samples were electrophoresed on a 10%T, 1.5%C polyacrylamide gel with 5% glycerol, in 0.5x TBE at room temperature, 150 Volts for 20 hours.

nucleotide changes underlying the variant sites were identified by direct sequencing (see next section).