In view of the LOH seen on chrom som e 5, and the absence of any data
p ro v id in g further localisation, candidate gene analysis w as carried out. The APC gene, at 5q21-22 is m utated in a num ber of adenocarcinom as, and as there have been no published m utation analysis studies of APC in DC, this gene w as a rational choice for further investigation. There w ere
no cases of rearran g em en t of APC using the cDNA probe FB54D. The
position of this probe w ithin the APC cDNA is show n in Figure 13. The APC cDNA w ith positions of m utations in FAP and colorectal cancer is show n in Figure 14.
SSCP w as used to look for m utations as it is a quick an d sensitive
m eth o d for detection of base p air changes (O rita et al, 1989). All the
p u b lish ed m utations seen in the cancers listed in the in tro d u ctio n w ere seen in exons 3-15, w ith the m ajority occurring in exon 15, b etw een
p rim er p airs 15b-i (using the nom enclature of G roden et a l 1991).
^ The number of tumours with LOH with any marker divided by the total number of informative tumours in that histopathological category.
^ The number of tumours with loss of all markers divided by the number with loss of any marker.
^ The number of tumours with loss of all markers on both amns divided by the number with loss anywhere on both 5p and 5q.
^ Serous papillary adenocarcinomas include serous papillary cystadenocarcinoma, serous
carcinoma and papillary carcinoma; Mucinous adenocarcinomas include mucinous cystadenocarcinoma. Cases of trisomy have been excluded from the totals.
TM l \ ) C J $ k O l O ) " ^ 0 0 ( O - k - L - & - L - L 4 c o r o CO ^ EXONS
S
FB54D
i
M
FB70B
o>
FB9A
::^
o*■
"
lO 00 o> CJl ro F ig u re 13 A c a r to o n o f th e A PC cDNAThere is an ORF of 8538 nucleotides. The predicted protein has 2843 amino acids. The position of cDNA probes is indicated by the black bars below the exon structure.
Figure 14. Mutations in A P C
The p o sitio n s of the g erm lin e (FAP) an d som atic (colon cancer)
m u tatio n s w ith in A P C are shown. The num ber refers to the num ber of
cases reported in the literature. It can be seen that the vast m ajority of all m utations occur in the first half of the gene.
0) n E 3 2 5 T 2 0 - - 1 5 -- 10 - - 5 -- L J C olon C a I I 1 4 I I I I " I o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o • ^ c o L O h - o ) - * - c o m t ^ o > T - c o i r ) r ^ o > 1— T— T— T— T— CM C \J C \J CM CM Codon Figure 14. M utations in APC
Therefore prim ers for exons 3-15i w ere used. Band shifts w ere seen using prim ers flanking exons 5,11 and 13. In exon 15 we noted band shifts w ith
exonic prim ers for exons denoted 15c,e, g and i in G roden et al. (1991).
These shifts w ere also seen in the lym phocyte DNA w h ere it w as available. All the ban d shifts seen w ere due to polym orphism s and are sum m arised in Table 8, along w ith all other published polym orphism s in APC . The frequency of the know n polym orphism s detected w as in k e e p in g w ith p re v io u s d e sc rip tio n s. A p re v io u s ly u n r e p o r te d polym orphism in exon 15e (TTG-TCG, codon 1129) w as noted by direct sequencing of a PGR p ro d u ct from norm al v o lu n teer DN A th a t h ad sh o w n a b a n d sh ift on SSCP (d ata n o t show n). In a d d itio n , th is polym orphism can be detected by digesting the PGR reaction w ith M bdll at 37°c. The b a n d s w ere revealed by ru n n in g the p ro d u c ts on an e th id iu m b ro m id e stain ed 2% agarose gel. If the p o ly m o rp h ism is present then the 225 bp fragm ent is digested to 195 + 30 bp. There is also a 100 b p constant fragm ent. This polym orphism in exon 15e w as detected in 1 /8 0 chrom osom es from OG patients and 1/8 0 chrom osom es from norm al Caucasian volunteers (Figure 19).
The polym orphism s w ere detected by direct sequencing (intron 5, exons 1 3 ,15c and 15g) and restriction digests of PCR reactions (exons 1 1 ,15e, 15g and 15i). The polym orphism in exon 15c could have been detected by BstNl, b u t this polym orphism w as sequenced directly. Examples of all the polym orphism s detected are show n in Figures 15-21.
For direct sequence of the band shift seen w ith intronic prim ers flanking exons 5 and 13, nested prim ers were designed:
5UP2: 5-TTTTCCTTACAAACAGATTTGACCAGA-3’ 13UP2: 5-TTATTGCAAGTGTTTTGAGGAA-3'
13RP2: 5'-TTTTAACTTCTAAAGCACATTCC-3' These prim ers w ere used in the sequencing reactions.
11 13 17
B
F ig u re 15 D N A p o ly m o rp h is m s in A P C exo n 5
A. SSCP of A P C e x o n 5 in tu m o u rs 11, 13, a n d 17. T he b a n d sh ift is s e e n w ith in tro n ic p rim e rs fla n k in g e x o n 5 in tu m o u r 13 o nly. C o n d itio n s : 6% p o ly a c r y la m id e g e l w ith 10% g ly c e ro l, r u n a t ro o m t e m p e r a t u r e o v e rn ig h t. NB b a n d sh ift n o t seen w ith 0% o r 5% glycerol.
B. T h e s e q u e n c e c h a n g e c o r r e s p o n d in g to th e b a n d s h ift is s h o w n . H e te ro z y g o s ity fo r G C G -G T G is se e n in b o th ly m p h o c y te a n d tu m o u r D N A for tu m o u r p a ir 13. N o c h a n g e is seen in n o rm a l a n d tu m o u r p a ir 11. ’^ p o ly m o rp h ism
41 42 47 48 50 51 53 61 64 67 4142474 85 0 51 5 3 616 4 67 - # -2 2 0 /2 0 0 1 5 4 /1 4 2 ♦ " 7 5
B
F ig u re 16 D N A p o ly m o rp h is m s in A P C ex o n 11 A . SSCP of A P C w ith p r im e r s fla n k in g ex o n 11. E x a m p le s of th e re s u lts s e e n w ith tu m o u r D N A s a m p le s 41-67 a re s h o w n . T h e to p b a n d is c o n s ta n t. A b ia lle lic p o ly m o r p h is m is r e v e a le d a n d th e a r r o w h e a d s in d ic a te d im in is h e d s ig n a l fro m th e lo st allele in tu m o u r s 41 a n d 48 (see F ig u re 10).B . A n e t h i d i u m b r o m i d e - s t a in e d 2% a g a r o s e g e l is s h o w n . T h is p o ly m o r p h is m (TA C -TA T, c o d o n 486) can b e d e te c te d b y Rs al d ig e s tio n of P C R p r o d u c ts o f e x o n 11. R s a l (GT A C) c le a v e s th e D N A a n d th e f r a g m e n t siz e s a re 86 a n d 129 b p . T he c h a n g e o f C to T r e s u lts in a u n c le a v e d fra g m e n t o f 215 b p . N o n -ra d io a c tiv e PC R a m p lific a tio n s fro m ly m p h o c y te c o m p a n io n s of th e tu m o u rs s h o w n in A are illu s tra te d T he alleles 'lo st ' in A can b e c learly seen. M o lecu lar w e ig h t size m a rk e rs a re s h o w n to th e rig h t of th e la st lane.
32 37 42 1 T I
G A T C
B
F ig u re 17 D N A p o ly m o rp h is m in A P C ex o n 13. A A n e x a m p le of th e p o ly m o r p h is m in e x o n 13 is s h o w n . T u m o u r 37 s h o w s a b a n d sh ift w h ic h is n o t se e n in th e fla n k in g tu m o u rs . B. T h e s e q u e n c e c h a n g e c o r r e s p o n d in g to th e b a n d s h ift s e e n in A is s h o w n . T his w a s also seen in th e p a r tn e r ly m p h o c y te D N A (n o t s h o w n ),4 2 9 0
G
A
C
1 2 3 4 5 6 7 8B
F ig u r e 18 D N A p o ly m o rp h is m in A P C ex o n 15cA SSCP of A P C w ith ex o n ic p r im e r s fla n k in g 15c. A b a n d s h ift is s e e n w ith tu m o u r sa m p le 90. B. T he se q u e n c e c o rre s p o n d in g to th e b a n d s h ift in A is s h o w n . L an es 1, 3, 5 a n d 7: w ild ty p e D N A P C R p ro d u c t. L a n e s 2, 4, 6 a n d 8: D N A fro m tu m o u r 90. p o ly m o r p h is m (C C A -T C A , c o d o n 870). T h e re is n o e v id e n c e of L O H . N o ly m p h o c y te D N A w a s a v a ila b le b u t as th e se q u e n c e c h a n g e is a k n o w n p o ly m o r p h is m it is a s s u m e d th a t th e s e q u e n c e c h a n g e w o u ld also b e p r e s e n t in th e g e rm line.
77t 79n79i
225
195
100
+ 77 79 79
T N TB
F ig u re 19 A n e w D N A p o ly m o rp h is m in A P C e x o n 15eA SSCP o f A P C w ith exon ic p rim e rs fla n k in g 15e. A b a n d s h ift is s e e n in tu m o u r 77.
B. A n e th id iu m b ro m id e sta in e d 2% a g a ro s e g el is s h o w n . A p re v io u s ly u n r e p o r te d p o ly m o r p h is m in e x o n 15e (TTG -TC G , c o d o n 1129) c a n b e d e te c te d b y d ig e s tin g th e PCR re a c tio n w ith M b o l l . W h e n th e r a r e allele is p re s e n t th e 225 b p fra g m e n t is d ig e ste d to 195 a n d 30 b p . T h e re is also a 100 b p c o n s ta n t fra g m e n t. L an e 1: lOObp la d d e r , w ith th e p o s itio n s of p o ly m o r p h ic b a n d s in d ic a te d to th e left. L a n e 2: P o s itiv e c o n tro l (th e n o r m a l v o lu n te e r in w h o m th e p o ly m o r p h is m w a s firs t n o te d ); L a n e 3: T u m o u r 77. T he s a m e d ig e s tio n p a tte r n w a s s e e n w ith 77 N ( d a ta n o t s h o w n ); L a n e s 4 a n d 5: L y m p h o c y te (79N ) a n d tu m o u r (79T) s a m p le s fro m A . N o d ig e stio n is seen.
77 7 8 8 0
78
80
n] 'Tj c 6 1 3» - 2 7 1* - 1 1 8» - F ig u r e 20 D N A p o ly m o rp h is m in A P C exon 15gA . SSCP of A P C w ith exonic p rim e rs fla n k in g ex o n 15g in tu m o u rs 77, 78 a n d 80. A b a n d sh ift is seen in tu m o u r 78. T his b a n d sh ift w a s seen w ith 0, 5 a n d 10% glycerol. T he w ild -ty p e b a n d p a tte r n is also seen.
B. Left: T h e re is n o se q u e n c e c h a n g e in tu m o u r 77. Right: T he s e q u e n c e
c h a n g e c o rre s p o n d in g to th e b a n d sh ift seen in tu m o u r 78 is sh o w n . T he w ild ty p e s e q u e n c e is also seen. T his c h a n g e w a s also o b s e rv e d in th e ly m p h o c y te D N A (n o t s h o w n ). p o ly m o r p h is m (G A A -C A A , c o d o n 1317).
C . D ig e s tio n o f th e 383 b p w ild -ty p e PC R p r o d u c t fro m e x o n 15g w ith
M b o l l p r o d u c e s fra g m e n ts of siz e s 92, 77, 64, 58, 56 a n d 3 6b p (th e se
f r a g m e n ts a re n o t in d iv id u a lly s e p a r a te d o n th is gel). If th e m u ta tio n s h o w n in A a n d B is p re s e n t th e n th e e n z y m e fails to cu t a t c o d o n 1317,