Proceso S4: Identificación y análisis de riesgos

mutation, three analytical instruments and a number of gel components were tested. The experiments involved PhastSystem™ using 20% polyacrylamide gel (PhastGel® Homogeneous 20) in combination with ‘Native’ or ‘DNA’ Buffer Strips, GenePhor™ using 12.5% and 15% polyacrylamide gel (GeneGel® Excel and GeneGel® Clean), ALF Express™ using 0.5x, 0.3x MDE® acrylamide gel and 0.5x gel with 5% glycerol. Several running times (200Vh, 250Vh, 300Vh, 350Vh and 400Vh for PhastSystem™ and 1 h 30 min, 2 h and 2 h 30 min for GenePhor™) and temperature conditions (5°C, 10°C, 15°C and 20°C) were employed. In total, 42 PhastGel™ (12 lanes of each run), 12 GeneGel® (24 lanes of each run) and 27 F-SSCP runs (20 lanes of each run) were used.

3,2,2 PGD strategy fo r P-thalassaemia, IVSI-110 mutation

Adding HUMTHOl primers (Kuliev et a l, 1998) (Table 3.2) to the outer

p-thalassaemia primers (El-Hashemite et a l, 1997) (Table 3.2) as a multiplex PCR

(Section 2.2.S.3) provides the advantage of linkage analysis, in order to confirm direct

mutation analysis from p-thalassaemia primers, in addition to contamination detection. The forward HUMTHOl primer was labelled with the blue fluorescent dye (Cy5®)

(Section 2.2.5.S) to allow analysis to be performed on an automated fluorescence

sequencer, the ALF Express™ (Section 2.2.7.2.1). The inner nested amplification reaction

(Section 2.2.S.4) was carried out using an aliquot of the outer amplification products as

the templates and the inner P-thalassaemia primers (El-Hashemite et a l, 1997) (Table

3.2). This resulted in the production of a fragment of 223 bp, 56 bp smaller than that

produced by the outer p-thalassaemia primers. The PCR products from the inner amplification were analysed by 2% agarose gel electrophoresis (Section 2.2.7.1) and

Chapter 3 Thalassaemias 103

SSCP analysis on GenePhor™ (Section 2,1,13,2). A control study comparing the

efficiency of two polymerase enzymes, SuperTaq® and AmpliTaq Gold™, was conducted.

3,2,3 PGD strategy fo r P-thalassaemia^ codon 41-42 mutation

The nature of the P-thalassaemia, codon 41-42 mutation is a 4 bp deletion (-TCTT) which can simply be identified using F-PCR (Section 2.2.5.5), The PGD

protocol for the p-thalassaemia codon 41-42 mutation was designed such that fragments from both p-thalassaemia and HUMTHOl loci (labelled in green, TET®, and blue,

6-FAM®, respectively) could be analysed at the same time on an automated fluorescence sequencer, the ABI Prism™310 (Section 2,2,1,2,2) following a multiplex amplification

(Section 2.2.5.3). The bthalwl primers (Table 3.2) were employed for mutation detection

giving a product size of 364 bp. The peak areas of the HUMTHOl alleles and the normal and mutant alleles of the P-globin gene of each sample were observed and scored for ADO and PA.

3,2,4 PGD strategy fo r a-thalassaemia, mutation

The a-thalassaemia, mutation is a large deletion defect within the a-globin gene cluster which removes both a l- and a2-globin genes. The traditional diagnostic method involves Southern blot which is not applicable for PGD. PCR amplification of the whole 20kb fragment in order to include the deletion is inefficient at the single cell level. Gap PCR (Section 2.2.S.6) is a wisely developed PCR based technique for molecule

analysis of such a large deletion while maintaining the amplification efficiency. In the analysis of the a-thalassaemia, mutation, the primers SI and S2 results in a PCR

Chapter 3 Thalassaemias 104

product of 287 bp for the normal allele and the primers SI and S3 generates a fragment of 194 bp for the mutant allele (Figure 3.6). The primers S2 and S3 were tagged with TEX'® (green) and HEX® (yellow/black) fluorescent dyes so that the normal and mutant alleles can be identified and distinguished on an ABI Prism"310. The polymorphic HUMTHOl primers, labelled with 6’FAM® (blue) fluorescent dye, were included as a multiplex reaction for contamination detection. The single step multiplex gap fluorescent PCR protocol was tested and optimised using single buccal cells of a heterozygote subject for the a-thalassaemia, mutation so that the amplification efficiency and ADO rates could be evaluated. The final optimised protocol was carried out on single human blastomeres donated for research.

10kb

a-globin gene complex

L— HYP

a-globin gene complex A Â with SEA deletion S11 I S3

Figure 3.6 a-Globin gene complex and a-thalassaemia SEA mutation with mapping of gap PCR primers. For a normal genotype, SI and S2 primers generate a PCR product of 287 bp, while SI and S3 primers result in an amplified fragment of 194 bp for the SEA mutation with 20kb deletion.

Chapter 3 Thalassaemias 105

3.2.5 Single cell sequencing

Single cell sequencing (Section 2.2.T.4) was applied to single cell PCR samples

that had been previously shown to have equal amplification of both alleles (SA); preferential amplification of the normal allele (PAN); preferential amplification of the mutant allele (PAM); ADO affecting the normal allele (ADON); ADO affecting the mutant allele (ADOM) for P-thalassaemia, IVSI-110 (substitution) and codon 41-42 (-TCTT frameshift) mutations (Section 3.2.2 and 3.2.3). Isolated human blastomeres,

derived from embryos donated for research, were also tested.

3.2.6 Single cell minisequencing (SNaPshoi'*)

Single cell PCR samples of heterozygote subjects with various P-globin gene mutations with known amplification characteristics, i.e. SA, ADOM, ADON, were processed with the minisequencing (SNaPshot""^ protocol (Section 1.2.1.S). The p-globin

gene mutations tested in this study involved P-thalassaemia IVSI-110 (Section 3.2.2), codon

41-42 (Section 3.3.2) and sickle. The ssIVSIllO, sscd4142 or sssickle minisequencing

primers were employed in the minisequencing reactions for the IVSI-110, codon 41-42 and sickle mutations, respectively (Table 3.3). The single cells from the sickle heterozygote

subject were amplified using the bthalw2 primers (Section 2.2.S.2 and Table 3.2). The

protocol for IVSI-110 mutation was applied to single human blastomeres donated for research.

Table 3.3 The details of the primers used for single cell minisequencing.

Primers Sequences (5 '-3 ') Location References

ssIVSIllO sscd4142 sssickle

5'-ACT GAG TCT CTC TOC CTA TT-3' 5 -CTA CCC TTG GAC CCA GAG GTT-3'

5 -GAC ACC ATG GTG CAC CTG ACT CCT G-3'

llp l5 .5 llp l5 .5 1 lpl5.5 OMIM OMIM OMIM

Chapter 3 Thalassaemias__________________________________________________________________________________________________________________106

3.3 Results

3,3.1 Mutation detection o f p-thalassaemia

During the experiments, problems with analytic instruments and techniques were experienced and corrected. Firstly, the silver staining quality of the PhastGel® was so pale that the band patterns could not be analysed. Formaldehyde and gluteraldehyde were found to be critical for silver staining, these reagents serve as developer and sensitiser respectively. The original manual silver staining protocol (Harvey et a l, 1995) excluded

the use of the glycerol solution and resulted in flaking of the GeneGel® surface within a few days, before a picture of the band pattern was taken. The introduction of the final glycerol solution step helped in gel preservation at room temperature for at least 2 months.

The amplified product of the IVSI-110 mutation using the inner p-thalassaemia primers could be identified on the Phast System™ using 20% PhastGel® and Native Buffer Strips with the conditions 15°C and 20°C, 350Vh (Figure 3.7). Using the

GenePhor™ for the analysis of the amplified products using the outer and inner P-thalassaemia primers, only the IVSI-110 mutation showed a different band pattern from the normal (Figure 3.8). The optimal condition was to use a 12.5% polyacrylamide gel at

5°C for 1 h 30 min. The reproducibility of this analysis in identifying the IVSI-110 mutation is shown in Figure 3.9.

Chapter 3 Thalassaemias 107