Sistema de riego de tuberías móviles de aluminio

N T (a) (b) G A T C c c T C G A C C G T T A G C T T G C T G A G A G T T T

S

Figure 3.8. Som atic m utation o f the A P C gene identifîed in the exon 15H am plicon in case COCA32. (a) SSCP and heteroduplex analysis. N and T denote DNA samples from normal and corresponding tumour tissues respectively. Both single strand variation (SS) and heteroduplexes (Het) were detected in the tumour, (b) Sequence analysis revealed a 29 bp deletion in the tumour DNA involving codons 1395-1405, which creates a stop codon immediately downstream. The sequence of the sense strand is shown.

Figure 3.9. N T 1 Horn (a) 5’ c; A T C

Norm al Tum eur

(b)

Figure 3.9. Detection o f somatic mutation in exon 15B amplicon o f A P C in case COCA34. (a) Heteroduplex analysis only of amplicon 15B, T and N denote tumour and corresponding normal DNA samples respectively. No single-stranded DNA mobility shift was detected in the tumour but heteroduplexes (Het) were observed, with a retarded mobility compared with homoduplex DNA (Hom). (b) Sequence analysis of exon 15B amplicon (antisense strand). A 2 bp deletion (TT) was identified in the tumour DNA sequence at codon 772-773 which leads to frameshift and a premature stop codon.

Figure 3.10.

N T N T

Het

]

Het

EXON 15E EXON 15F

Figure 3.10. SSCP and heteroduplex analysis o f am plicons 15E and 15F o f APC in co rresp o n d in g tu m o u r and n o rm a l DNA from case COCA39. Heteroduplex shifts were detected in the tumour DNA in both amplicons 15E and

15F (Het=heteroduplexes; SS=single strands).

However no sequence alteration was identified in either amplicon in the tumour DNA.

Figures 3.11 to 3.13 illustrate somatic variants detected in amplicon 15G of

APC. As shown in figure 3.1 la, a similar variant single-stranded DNA pattern was detected in tumour DNA from cases COCA26 and COCA28. Sequence analysis (figure 3.11b) demonstrated the novel conformers to be the result of an A to T transversion resulting in a nonsense mutation of arginine (AGA) to a stop codon (TGA) at codon 1331; truncation of the APC protein would be predicted. Likewise, a similar variant single strand band pattern was detected in tumour DNA from cases C0CA7 and COCA23 as illustrated in figure 3.12. The electrophoretic variation was characterized in the tumour DNA from both cases as being the result of a single base change from guanine to thymine at the first nucleotide of codon 1322, causing a nonsense mutation from glutamic acid (GAA) to a stop codon (TAA); a change also predicted to be truncating.

Figure 3.13a depicts SSCP and heteroduplex analysis of amplicon 15G in DNA from cases C0CA2, COCA40, and COCA42. Tumour DNA from all three cases did not exhibit single-stranded DNA variation but a heteroduplex DNA variant of a similar mobility. Figure 3.13b shows sequence analysis of amplicon 15G in tumour and normal DNA from case COCA2. A 5 bp deletion (AAAGA) was detected at codon 1307-1311. This change caused a frameshift that generated a new stop codon at immediately downstream. An identical sequence alteration was observed in tumour DNA of cases COCA40 and COCA42.

SSCP and heteroduplex analysis identified variants in APC amplicon 15H in tumour DNA from three more cases as shown in figures 3.14, 3.15, and 3.16. Firstly, in case C0CA9 a single-stranded DNA conformer of differing mobility was detected in the tumour DNA in addition to the wild-type single strand DNA pattern (figure 3.14a). Heteroduplexes were not detected. Subsequent sequence analysis revealed a single base substitution of an A residue for a G residue in the tumour DNA, causing a missense mutation from valine (GTT) to isoleucine (ATT) at codon 1405 (figure 3.14b).

Figure 3.15 shows heteroduplex analysis of the exon 15H amplicon in tumour and normal DNA from case COCA24. A heteroduplex variant migrating at a slightly slower rate than the homoduplex DNA molecule was detected in the tumour DNA. The variant was characterized as a 2 bp deletion, of either AG or GA in the sequence AAAAGAGAGAGAGT at codon 1462-1465, that causes a frameshift creating an

early stop codon immediately downstream. In tumour DNA from case COCAS 8, again a heteroduplex shift but no single strand mobility shift was observed (figure 3.16a). Sequencing of amplicon 15H in the tumour DNA revealed a 4 bp insertion at codon 1395 (figure 3.16b). This sequence change also results in frameshift and the formation of a termination codon at immediately downstream.

Finally, as shown in figure 3.17, a somatic variant was detected in case COCAS in amplicon 151 of APC. Single-stranded DNA variants were not seen in the tumour DNA. Double-stranded homoduplex DNA was retained on the gel however, and a homoduplex variant of greater mobility than the wild-type homoduplex DNA molecule was detected. Sequencing demonstrated this variant to be the result of a somatic deletion of an A residue in the sequence GAAAGT at codon 1494-1495. Once again this mutation led to frameshift and created a new stop codon 33 nucleotides downstream that is predicted to cause truncation of the A P C gene product.

As described in table 3.2, common polymorphisms were detected in amplicons 151 and 15J, these will be more fully discussed in section 3.1.2. A total of 19 somatic APC mutations were identified in 43 colorectal carcinomas as described in table 3.1. Nine of them (47%) were point mutations; seven of these were nonsense mutations resulting in truncation of the gene product whereas only one was a missense mutation. The remaining point mutation occurred at a splice site and could lead to frameshift (case COCA35). Five of the nine point mutations were transitions and four were transversions. Four of the transitions were G:C to A:T changes, of which two occurred at CpG dinucleotide pairs and two occurred at CpA dinucleotide pairs. One transition was an A:T to G:C change. Of the four transversions, two were G:C to T:A changes and two were A:T to T:A changes. Eight of the nineteen somatic mutations were deletions of 1 to 29 bp and two were insertions of 4 bp. All of the deletions and insertions produced a frameshift creating new termination codons at downstream. Thus the great majority of somatic mutations observed (18 of 19, 95%) were predicted to lead to truncation of the APC product.

Three of the mutations characterized were found in more than one case: the 5 bp deletion at codon 1307-1311 was observed in three cases, the nonsense mutation at codon 1322 (Glu to stop) in two cases, and the nonsense mutation at codon 1331 (Arg to stop) in two cases. The majority of somatic mutations were located in exon 15 of

APC. Seventy-four percent (14 of 19) fell into the region of this exon, between codons 1286 and 1513, designated as 'mutation cluster region' (Miyoshi et a l,

Figure 3.1 1 2 3 4 SS (a) G A T C 5’ 3' Normal (b) G A T C 5 ’ C T A/T G A A C 3'

i

i

Figure 3.11. D etection o f som atic m utation in exon 15G am plicon o f A P C in cases COCA26 and COCA28. (a) SSCP analysis. Lanes 1 and 2 represent SSCP of corresponding tumour and normal DNA respectively from case COCA26, lanes 3 and 4 represent tumour and normal DNA from case COCA28. In addition to the w ild-type single strand (SS) band pattern, tum our DNA from both cases demonstrate a similar variant single-stranded DNA pattern, as indicated by the arrows, (b) Sequencing of 15G amplicon (antisense strand) in tumour and normal DNA from case COCA26. A single base substitution of T for A causes a change from Arg (AGA) to stop (TGA) at codon 1331. An identical sequence change was identified in tumour DNA from COCA28.

Figure 3.12. 1 2 3 4 SS (a) G A T C G A T C 5' 5' ^ A A-^ ^ G G C C G GAI A A A A G G

Kî

Normal Tum our

(b)

Figure 3.12. Detection o f som atic m utation in exon 15G am plicon o f APC in cases COCA7 and COCA23. (a) SSCP analysis. Lanes 1 and 2 represent SSCP of corresponding normal and tumour DNA respectively from case C 0C A 7, lanes 3 and 4 represent normal and tumour DNA respectively from case COCA23. Novel single-stranded (SS) DNA conformers of a similar mobility were observed in the tumour DNA from both cases (see arrow), (b) Sequence analysis of exon 15G amplicon in tumour and normal DNA from case COCA23. A G to T transversion was identified in the tumour resulting in a change from glutamic acid (GAA) to a stop codon (TAA) at codon 1322. The sequence of the antisense strand is shown.

Figure 3.13.

PN T N T N

Het