CONCLUSIONES

150 H H 4B : DR5-4M / DR5-4 / / i140 (9 repeats) 450 HH 40 : DR5-4*4 / DR5-4 / / 154 oj- H H 78 : DR5-7*7 / DR5-7 / / i140 (9) .149 (12) HH 70 : DR5-7*7 / DR5-7 / /

Figure 4.7 DM and APOC2 results multiplex amplified from single buccal cells analysed on the ABI Prism ^*310 using GeneScan® analysis. Lanes 4B and 40 are the analyses of APOC2 (lane 4B, blue) and DM (lane 40, green) genes from the same single buccal cell sample, show AF of AP0C2 fragment and ADOL of DM gene. The results from another cell, lanes 7B and 70 show ADOS of AP0C2 fragment and SA of the DM gene. Split peak or Plus-A problem is reduced dramatically after the modification of the PCR protocol. The x and y axes are base and peak height units, respectively.

The reproducibility of the analysis results from the ABI Prism'^310 was tested by programming the machine to analyse the same multiplex PCR product ten times. The standard deviations of the peak areas of four alleles (two of DM and two of the AP0C2 genes) from ten analyses are between 2.60% and 6.08%, and those of the ratios between two alleles of the same gene are between 3.52 and 5.88%. Therefore, true preferential amplification in this study was defined as the samples having differences between both alleles of more than 10%.

The multiplex amplified products from single cells were each tagged with two different fluorochromes using labelled primers. This allowed analysis to be performed on

Chapter 4 Myotonic Dystrophy__________________________________________________________ 172

an ALF Express™ and also an ABI Prism™310. Consequently it was possible to compare the performance of these two pieces of equipment. DM fragments were labelled with the green fluorescent dye (TET®) (forward primer) and a blue dye, Cy5® (reverse primer). AP0C2 and D21S1414 fragments were both labelled with the blue fluorescent dye 6-FAM® (forward primers) and Cy5® (reverse primers). The analyses of the same samples from both sequencers showed corresponding results. However, the ABI Prism™310 provided a flatter baseline and more precise fragment size results, while the ALF Express^” gave quicker results if more than 4 samples were analysed.

Analyses were carried out on 50 single buccal cells from normal heterozygote subjects. Amplification using DM primers alone provided results from 94% of cells with an ADO rate of 12.8% (Table 4.3). After these promising results, 100 single cell PCRs using a combination of DM and AP0C2 primers (protocol 1) were attempted. These showed amplification in 95% of samples for both DM and AP0C2 loci; ADO was observed in 9.5% of DM amplifications and 10.5% of AP0C2 amplifications. Multiplex PCR analysis of 90 single buccal cells from DM carriers and affected patients (where only the normal allele can be detected) revealed 82.2% amplification efficiency for the DM primers. The reduced amplification efficiency observed when the carrier cells were tested could be due to the fact that all instances of ADO affecting the normal allele will resemble amplification failure. Multiplex amplification using DM and D2IS 1414 primers (protocol 2) on 50 normal heterozygous single buccal cells demonstrated 98% amplification efficiency for both sets of primers. The DM gene displayed a 4.1% ADO rate, while for D21S1414 it was 14.3%. The studies on single buccal cells indicate that the addition of a polymorphic marker to the DM gene analysis as multiplex PCR does not adversely affect the amplification efficiency or ADO rates.

Chapter 4 Myotonic Dystrophy 173

Table 4.3 Summarised data showing amplification efficiencies and ADO rates of DM, AP0C2 and D21S1414 loci from single buccal cells using singleplex (DM only) and multiplex (DM&AP0C2 and DM&D21S1414) PCR.

Study DM A P 0 C 2 D 2 1 S 1 4 1 4 amplification efficiency ADO amplification efficiency ADO amplification efficiency ADO DM only 94% 12.8% (4 7 /5 0 ) (6 /4 7 ) D M & A P 0 C 2 95% 9.5% 95% 10.5% (protocol 1) (9 5 /1 0 0 ) (9 /9 5 ) (9 5 /1 0 0 ) (1 0 /9 5 ) D M & D 21 S 14 14 98% 4.1% 98% 14.3% (protocol 2) (4 9 /5 0 ) (2/4 9 ) (4 9 /5 0 ) (7 /4 9 )

Triplex PCR using DM, AP0C2 and D21S1414 primers, which would detect an even greater number of contaminants, is also possible as shown in Figure 4.8.

90 120 150 180 210 240 270 300 330 380 390 600. 450. 300. 150. 0 5850 4680 3510 2340 1170 0

two AP0C2 alleles two D21S1414 alleles with preferential_{amplification of the shorter alleles h o rte r allele}

l a a 48 :C3ma14W8-6 /

^ ^ tw o normal DM alleles

^ w i t h preferential amplification of the shorter allele

U L ________________

H H 4G :C 3m a14W S-6/

Figure 4.8 DM, AP0C2 and D21S1414 results triplex amplified from a single buccal cell of a heterozygote subject for the 3 loci analysed on ABI Prism^'^310 using GeneScan analysis. Lane 4B (blue) shows 2 AP0C2 alleles and 2 D21S1414 alleles, lane 40 (green) shows two normal DM alleles. The x and y axes are base and peak height units, respectively.

Chapter 4 M yotonic Dystrophy 174

In both studies, there was no contamination in any of the final wash drop blanks, lysis-buffer-only negative controls or PCR reaction-mixture-only negative controls. One wash drop blank was taken for every 10 single buccal cells and one for every single blastomere, a lysis-buffer-only negative control and a PCR reaction-mixture-only negative control were taken for every experiment.

Multiplex F-PCR using DM and AP0C2 primers was conducted on 31 spare single blastomeres donated for research and showed 93.5% (29/31) amplification efficiency for each locus. ADO rates were estimated to be 6.9% (2/29) and 10.3% (3/29) for DM and AP0C2, respectively. Multiplex fluorescent analysis for DM and D2IS 1414 loci was carried out on 23 single blastomeres giving an amplification efficiency of 95.7% (22/23) for each locus. ADO rates for DM were 4.5% (1/22) while for D21S1414 they were 22.7% (5/22). However, chromosomal mosaicism can make ADO appear higher in this cell type (Delhanty et al, 1997). All final wash drop blanks, lysis-buffer-only

negative controls and PCR reaction-mixture-only negative controls were negative for DNA contamination.

4 3 ,2 Work up fo r D M PGD cases

PGD workup for two families, ‘A’ and ‘B’ (Table 4.4), at risk of having a

congenital DM child was performed using DNAs extracted from blood and buccal cell samples from the members of both families. The 36-year old carrier mother, ‘Am’, of the family ‘A’ experienced two spontaneous miscarriages and two terminations of pregnancy following positive prenatal diagnosis of DM after the delivery of a congenital DM baby. The healthy father, ‘A f , of this family was 38 years old, and the congenital affected son

Chapter 4 M yotonic Dystrophy 175

was 8 years old with a history of operations correcting talipes of both feet. The carrier mother, ‘Bm’, of family ‘B’, whose father and brother were mildly affected, was 32 years old. She delivered one congenital affected baby and had two terminations of pregnancy after positive chorionic villous sampling for DM. The father ‘B f , of this family was 34 years old and healthy, and the congenital DM son was 9 years old.

Table 4.4 Summarised details of families ‘A’ and ‘B’.

Family ‘A’ Family ‘B’

Carrier Mother age Father age Children Obstetric history Family history mother 35 years old 38 years old 1 congenital DM son, 8 years old 2 spontaneous miscarriages 2 TOP following positive PND

mother 32 years old 34 years old 1 congenital DM son,

9 years old

2 TOP following positive PND

affected father and brother DM = myotonic dystrophy, TOP = termination of pregnancy, PND = prenatal diagnosis

The results of F-PCR analyses and the numbers of CTG repeat in DM and AC repeats in AP0C2 for each member of the two families are shown in Figures 4.9-4.12.

There is only one normal allele (peak) of the DM gene of ‘Am’, ‘Ac’, ‘Bm’ and ‘Be’, shown in Figure 4.9 and 4.10, because the mutant CTG expansion is longer than 500 bp

and cannot be seen by the GeneScan® protocol. The expected genotypes of the DM and AP0C2 genes in the offspring of both families are demonstrated in Figure 4.10 and 4.12.

Chapter 4 Myotonic Dystrophy 176